Regulators of the hedgehog pathway, compositions and uses related thereto

ABSTRACT

The present invention makes available, inter alia, methods and reagents for modulating smoothened-dependent pathway activation. In certain embodiments, the subject methods can be used to counteract the phenotypic effects of unwanted activation of a hedgehog pathway, such as resulting from hedgehog gain-of-function, ptc loss-of-function or smoothened gain-of-function mutations.

This application is a continuation U.S. application Ser. No. 12/079,776,filed Mar. 28, 2008, which is a continuation of U.S. application Ser.No. 11/338,503, filed Jan. 23, 2006, now U.S. Pat. No. 7,476,661, whichis a continuation of U.S. application Ser. No. 09/688,076, filed Oct.13, 2000, now U.S. Pat. No. 7,098,196, which claims the benefit of U.S.Provisional Application No. 60/159,215, filed Oct. 13, 1999, and No.60/229,273, filed Aug. 30, 2000, the specifications of each of which arehereby incorporated by reference in their entirety; and this applicationalso claims the benefit of U.S. application Ser. No. 13/364,161, filedFeb. 1, 2012, which is a continuation of U.S. application Ser. No.12/652,134, filed on Jan. 5, 2010, which is a continuation of U.S.application Ser. No. 11/270,984, filed on Nov. 11, 2005, now abandoned,which is a divisional of U.S. application Ser. No. 09/708,964, filed onNov. 8, 2000, now U.S. Pat. No. 7,291,626, which is a continuation ofU.S. application Ser. No. 09/685,244, filed on Oct. 10, 2000, nowabandoned, which is a continuation-in-part of PCT Application No. US99/07811, filed on Apr. 9, 1999, which claims the benefit of U.S.Provisional Application Nos. 60/081,186, filed on Apr. 9, 1998, and60/081,263, filed on Apr. 9, 1998, and which is a continuation-in-partof U.S. application Ser. No. 09/090,622, filed on Jun. 4, 1998, now U.S.Pat. No. 6,432,970, which also claims the benefit of U.S. ProvisionalApplication No. 60/081,186, filed on Apr. 9, 1998; and is related toU.S. Ser. No. 11/894,712, filed on Aug. 20, 2007, now U.S. Pat. No.8,097,632, and U.S. Ser. No. 09/708,974, filed on Nov. 8, 2000, now U.S.Pat. No. 6,867,216.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinsic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diverse cell typesduring tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,(1992) Cell 68: 257-270). The effects of developmental cell interactionsare varied. Typically, responding cells are diverted from one route ofcell differentiation to another by inducing cells that differ from boththe uninduced and induced states of the responding cells (inductions).Sometimes cells induce their neighbors to differentiate like themselves(homeogenetic induction); in other cases a cell inhibits its neighborsfrom differentiating like itself. Cell interactions in early developmentmay be sequential, such that an initial induction between two cell typesleads to a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation (J. B. Gurdon (1992) Cell 68:185-199).

Members of the Hedgehog family of signaling molecules mediate manyimportant short- and long-range patterning processes during invertebrateand vertebrate development. In the fly, a single hedgehog gene regulatessegmental and imaginal disc patterning. In contrast, in vertebrates, ahedgehog gene family is involved in the control of left-right asymmetry,polarity in the CNS, somites and limb, organogenesis, chondrogenesis andspermatogenesis.

The first hedgehog gene was identified by a genetic screen in thefruitfly Drosophila melanogaster (Niisslein-Volhard, C. and Wieschaus,E. (1980) Nature 287, 795-801). This screen identified a number ofmutations affecting embryonic and larval development. In 1992 and 1993,the molecular nature of the Drosophila hedgehog (hh) gene was reported(C. F., Lee et al. (1992) Cell 71, 33-50), and since then, severalhedgehog homologues have been isolated from various vertebrate species.While only one hedgehog gene has been found in Drosophila and otherinvertebrates, multiple Hedgehog genes are present in vertebrates.

The vertebrate family of hedgehog genes includes at least four members,e.g., paralogs of the single drosophila hedgehog gene. Exemplaryhedgehog genes and proteins are described in PCT publications WO95/18856 and WO 96/17924. Three of these members, herein referred to asDesert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh),apparently exist in all vertebrates, including fish, birds, and mammals.A fourth member, herein referred to as tiggy-winkle hedgehog (Twhh),appears specific to fish. Desert hedgehog (Dhh) is expressed principallyin the testes, both in mouse embryonic development and in the adultrodent and human; Indian hedgehog (Ihh) is involved in bone developmentduring embryogenesis and in bone formation in the adult; and, Shh, whichas described above, is primarily involved in morphogenic andneuroinductive activities. Given the critical inductive roles ofhedgehog polypeptides in the development and maintenance of vertebrateorgans, the identification of hedgehog interacting proteins is ofparamount significance in both clinical and research contexts.

The various Hedgehog proteins consist of a signal peptide, a highlyconserved N-terminal region, and a more divergent C-terminal domain. Inaddition to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev.2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353),Hedgehog precursor proteins undergo an internal autoproteolytic cleavagewhich depends on conserved sequences in the C-terminal portion (Lee etal. (1994) Science 266:1528-1537; Porter et al. (1995) Nature374:363-366). This autocleavage leads to a 19 kD N-terminal peptide anda C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Chang et al.(1994) supra; Lee et al. (1994) supra; Bumcrot, D. A., et al. (1995)Mol. Cell. Biol. 15:2294-2303; Porter et al. (1995) supra; Ekker, S. C.et al. (1995) Curr. Biol. 5:944-955; Lai, C. J. et al. (1995)Development 121:2349-2360). The N-terminal peptide stays tightlyassociated with the surface of cells in which it was synthesized, whilethe C-terminal peptide is freely diffusible both in vitro and in vivo(Porter et al. (1995) Nature 374:363; Lee et al. (1994) supra; Bumcrotet al. (1995) supra; Marti, E. et al. (1995) Development 121:2537-2547;Roelink, H. et al. (1995) Cell 81:445-455). Interestingly, cell surfaceretention of the N-terminal peptide is dependent on autocleavage, as atruncated form of HH encoded by an RNA which terminates precisely at thenormal position of internal cleavage is diffusible in vitro (Porter etal. (1995) supra) and in vivo (Porter, J. A. et al. (1996) Cell 86,21-34). Biochemical studies have shown that the autoproteolytic cleavageof the HH precursor protein proceeds through an internal thioesterintermediate which subsequently is cleaved in a nucleophilicsubstitution. It is likely that the nucleophile is a small lipophilicmolecule which becomes covalently bound to the C-terminal end of theN-peptide (Porter et al. (1996) supra), tethering it to the cellsurface. The biological implications are profound. As a result of thetethering, a high local concentration of N-terminal Hedgehog peptide isgenerated on the surface of the Hedgehog producing cells. It is thisN-terminal peptide which is both necessary and sufficient for short- andlong-range Hedgehog signaling activities in Drosophila and vertebrates(Porter et al. (1995) supra; Ekker et al. (1995) supra; Lai et al.(1995) supra; Roelink, H. et al. (1995) Cell 81:445-455; Porter et al.(1996) supra; Fietz, M. J. et al. (1995) Curr. Biol. 5:643-651; Fan,C.-M. et al. (1995) Cell 81:457-465; Marti, E., et al. (1995) Nature375:322-325; Lopez-Martinez et al. (1995) Curr. Biol 5:791-795; Ekker,S. C. et al. (1995) Development 121:2337-2347; Forbes, A. J. et al.(1996) Development 122:1125-1135).

HH has been implicated in short- and long-range patterning processes atvarious sites during Drosophila development. In the establishment ofsegment polarity in early embryos, it has short-range effects whichappear to be directly mediated, while in the patterning of the imaginaldiscs, it induces long range effects via the induction of secondarysignals.

In vertebrates, several hedgehog genes have been cloned in the past fewyears. Of these genes, Shh has received most of the experimentalattention, as it is expressed in different organizing centers which arethe sources of signals that pattern neighboring tissues. Recent evidenceindicates that Shh is involved in these interactions.

The expression of Shh starts shortly after the onset of gastrulation inthe presumptive midline mesoderm, the node in the mouse (Chang et al.(1994) supra; Echelard, Y. et al. (1993) Cell 75:1417-1430), the rat(Roelink, H. et al. (1994) Cell 76:761-775) and the chick (Riddle, R. D.et al. (1993) Cell 75:1401-1416), and the shield in the zebrafish (Ekkeret al. (1995) supra; Krauss, S. et al. (1993) Cell 75:1431-1444). Inchick embyros, the Shh expression pattern in the node develops aleft-right asymmetry, which appears to be responsible for the left-rightsitus of the heart (Levin, M. et al. (1995) Cell 82:803-814).

In the CNS, Shh from the notochord and the floorplate appears to induceventral cell fates. When ectopically expressed, Shh leads to aventralization of large regions of the mid- and hindbrain in mouse(Echelard et al. (1993) supra; Goodrich, L. V. et al. (1996) Genes Dev.10:301-312), Xenopus (Roelink, H. et al. (1994) supra; Ruiz i Altaba, A.et al. (1995) Mol. Cell. Neurosci. 6:106-121), and zebrafish (Ekker etal. (1995) supra; Krauss et al. (1993) supra; Hammerschmidt, M., et al.(1996) Genes Dev. 10:647-658). In explants of intermediate neuroectodermat spinal cord levels, Shh protein induces floorplate and motor neurondevelopment with distinct concentration thresholds, floor plate at highand motor neurons at lower concentrations (Roelink et al. (1995) supra;Marti et al. (1995) supra; Tanabe, Y. et al. (1995) Curr. Biol.5:651-658). Moreover, antibody blocking suggests that Shh produced bythe notochord is required for notochord-mediated induction of motorneuron fates (Marti et al. (1995) supra). Thus, high concentration ofShh on the surface of Shh-producing midline cells appears to account forthe contact-mediated induction of floorplate observed in vitro (Placzek,M. et al. (1993) Development 117:205-218), and the midline positioningof the floorplate immediately above the notochord in vivo. Lowerconcentrations of Shh released from the notochord and the floorplatepresumably induce motor neurons at more distant ventrolateral regions ina process that has been shown to be contact-independent in vitro(Yamada, T. et al. (1993) Cell 73:673-686). In explants taken atmidbrain and forebrain levels, Shh also induces the appropriateventrolateral neuronal cell types, dopaminergic (Heynes, M. et al.(1995) Neuron 15:35-44; Wang, M. Z. et al. (1995) Nature Med.1:1184-1188) and cholinergic (Ericson, J. et al. (1995) Cell 81:747-756)precursors, respectively, indicating that Shh is a common inducer ofventral specification over the entire length of the CNS. Theseobservations raise a question as to how the differential response to Shhis regulated at particular anteroposterior positions.

Shh from the midline also patterns the paraxial regions of thevertebrate embryo, the somites in the trunk (Fan et al. (1995) supra)and the head mesenchyme rostral of the somites (Hammerschmidt et al.(1996) supra). In chick and mouse paraxial mesoderm explants, Shhpromotes the expression of sclerotome specific markers like Pax1 andTwist, at the expense of the dermamyotomal marker Pax3. Moreover, filterbarrier experiments suggest that Shh mediates the induction of thesclerotome directly rather than by activation of a secondary signalingmechanism (Fan, C.-M. and Tessier-Lavigne, M. (1994) Cell 79,1175-1186).

Shh also induces myotomal gene expression (Hammerschmidt et al. (1996)supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173; Miinsterberg, A.E. et al. (1995) Genes Dev. 9:2911-2922; Weinberg, E. S. et al. (1996)Development 122:271-280), although recent experiments indicate thatmembers of the WNT family, vertebrate homologues of Drosophila wingless,are required in concert (Munsterberg et al. (1995) supra). Puzzlingly,myotomal induction in chicks requires higher Shh concentrations than theinduction of sclerotomal markers (Munsterberg et al. (1995) supra),although the sclerotome originates from somitic cells positioned muchcloser to the notochord. Similar results were obtained in the zebrafish,where high concentrations of Hedgehog induce myotomal and represssclerotomal marker gene expression (Hammerschmidt et al. (1996) supra).In contrast to amniotes, however, these observations are consistent withthe architecture of the fish embryo, as here, the myotome is thepredominant and more axial component of the somites. Thus, modulation ofShh signaling and the acquisition of new signaling factors may havemodified the somite structure during vertebrate evolution.

In the vertebrate limb buds, a subset of posterior mesenchymal cells,the “Zone of polarizing activity” (ZPA), regulates anteroposterior digitidentity (reviewed in Honig, L. S. (1981) Nature 291:72-73). Ectopicexpression of Shh or application of beads soaked in Shh peptide mimicsthe effect of anterior ZPA grafts, generating a mirror image duplicationof digits (Chang et al. (1994) supra; Lopez-Martinez et al. (1995)supra; Riddle et al. (1993) supra) (FIG. 2g ). Thus, digit identityappears to depend primarily on Shh concentration, although it ispossible that other signals may relay this information over thesubstantial distances that appear to be required for AP patterning(100-150 μm). Similar to the interaction of HH and DPP in the Drosophilaimaginal discs, Shh in the vertebrate limb bud activates the expressionof Bmp2 (Francis, P. H. et al. (1994) Development 120:209-218), a dpphomologue. However, unlike DPP in Drosophila, Bmp2 fails to mimic thepolarizing effect of Shh upon ectopic application in the chick limb bud(Francis et al. (1994) supra). In addition to anteroposteriorpatterning, Shh also appears to be involved in the regulation of theproximodistal outgrowth of the limbs by inducing the synthesis of thefibroblast growth factor FGF4 in the posterior apical ectodermal ridge(Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, L. et al. (1994)Nature 371:609-612).

The close relationship between Hedgehog proteins and BMPs is likely tohave been conserved at many, but probably not all sites of vertebrateHedgehog expression. For example, in the chick hindgut, Shh has beenshown to induce the expression of Bmp4, another vertebrate dpp homologue(Roberts, D. J. et al. (1995) Development 121:3163-3174). Furthermore,Shh and Bmp2, 4, or 6 show a striking correlation in their expression inepithelial and mesenchymal cells of the stomach, the urogential system,the lung, the tooth buds and the hair follicles (Bitgood, M. J. andMcMahon, A. P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of thetwo other mouse Hedgehog genes, is expressed adjacent to Bmp expressingcells in the gut and developing cartilage (Bitgood and McMahon (1995)supra).

A major function of hedgehog in the Drosophila embryo is the maintenanceof wg transcription at the boundary of each segmental unit (Hidalgo andIngham, (1990) Development 110:291-302); from here, Wg protein diffusesacross the segment to specify the character of the ectodermal cells thatsecrete the larval cuticle (Lawrence et al., (1996) Development122:4095-4103). Like hh, mutations in three other segment polarity genessmoothened (smo), fused (fu) and cubitus interruptus (ci) eliminate wgtranscription at parasegmental borders (Forbes et al., (1993)Development Suppl. 115-124; Ingham, (1993) Nature 366:560-562; Préat etal., (1993) Genetics 135:1047-1062; and van den Heuvel et al. (1996)Nature 382:547-551); by contrast, mutation of a fourth gene, patched(ptc), leads to the derepression of wg (Ingham et al., (1991) Nature353:184-187; and Martinez Arias et al., (1988) Development 103:157-170).By making double mutant combinations between ptc and the other genes, itwas established that smo, fu and ci all act downstream of ptc toactivate wg transcription (Forbes et al., (1993) supra; Hooper (1994)Nature 372:461-464) whilst, on the other hand, transcription of wgbecomes independent of hh in the absence of ptc (Ingham and Hidalgo(1993) Development 117:283-291). These findings suggest a simple pathwaywhereby hh acts to antagonize the activity of ptc which in turnantagonizes the activity of smo, fu and ci. The universality of thispathway subsequently has been established both in Drosophila, where ptc,smo, fu and ci mediate the activity of Hh in all processes studied todate (Ma et al., (1993) Cell 75:927-938); Chen et al. (1996) Cell87:553-563; Forbes et al., (1996) Development 122:3283-3294;Sanchez-Herrero et al. (1996) Mech. Dev. 55:159-170; Strutt et al.(1997) Development 124:3233-3240), and in vertebrates, where homologuesof ptc, smo and ci have been identified and implicated in processesmediated by one or other of the Hh family proteins (Concordet et al.,(1996) Development 122:2835-2846; Goodrich et al., supra; Marigo et al.,(1996) Dev. Biol. 180:273-283; Stone et al. (1996) Nature 384:129-134;Hynes et al. (1997) Neuron 19:15-26; and Quirk et al. (1997) Cold SpringHarbor Symp. Quant. Biol. 62:217-226).

Patched was originally identified in Drosophila as a segment polaritygene, one of a group of developmental genes that affect celldifferentiation within the individual segments that occur in ahomologous series along the anterior-posterior axis of the embryo. SeeHooper, J. E. et al. (1989) Cell 59:751; and Nakano, Y. et al. (1989)Nature 341:508. Patterns of expression of the vertebrate homologue ofpatched suggest its involvement in the development of neural tube,skeleton, limbs, craniofacial structure, and skin.

Another protein involved in hedgehog signaling emerged with thediscovery that smoothened also encodes a transmembrane protein that is amember of the 7 transmembrane receptor (7™) family (Alcedo et al. (1996)Cell 86:221-232; van den Heuvel et al. supra). Human homologs of smohave been identified. See, for example, Stone et al. (1996) Nature384:129-134, and GenBank accession U84401. In vitro binding assays havefailed to detect any physical interaction between vertebrate Smo and Hhproteins (Stone et al., supra) whereas, under the same conditions,vertebrate Ptc binds the Sonic hedgehog (Shh) protein with relativelyhigh affinity (Marigo et al. (1996) Nature 384:176-179; Stone et al.,supra). Recently, it has been reported that activating smoothenedmutations occur in sporadic basal cell carcinoma, Xie et al. (1998)Nature 391: 90-2, and primitive neuroectodermal tumors of the centralnervous system, Reifenberger et al. (1998) Cancer Res 58: 1798-803.

The findings in the art suggest that Hh acts by binding to Ptc, therebyreleasing an inhibitory effect of Ptc on Smo. Since Ptc and Smo are bothtransmembrane proteins, a proposed scenario is that they physicallyassociate to form a receptor complex, though indirect mechanisms ofaction are also plausible. The derepression of Smo from Ptc inhibitionmost likely involves a conformational change in Smo. It is, however,important to remember that Ptc is not essential for Smo's activity,since Smo becomes constitutively activated in the complete absence ofPtc protein (Alcedo et al., supra; Quirk et al., supra).

It follows from the model that at least some loss-of-function mutationsin ptc should act by disrupting binding to Smo. The discovery thatmutations in the human ptc homolog are widespread in basal cellcarcinomas (BCCs) (Hahn et al. (1996) Cell 85:841-851; Johnson et al.(1996) Science 272:1668-1671) has provided a major stimulation for theanalysis of Ptc/Smo function as well as an abundant source ofloss-of-function mutations. Many tumour-derived alleles of human ptchave now been sequenced, with the majority of the mutationscharacterized being due to premature termination of the coding region(Chidambaram et al. (1996) Cancer Res. 56:4599-4601; Wicking et al.,(1997) Am. J. Hum. Genet. 60:21-26).

Disruption of Smo-Ptc binding could also be caused by mutations in smo;in contrast to ptc mutations, these should be dominantly acting (sincethey would lead to constitutive activity of the mutant protein). Recentstudies of human BCCs have identified activating mutation(s) in Smo andappear to be responsible for the transformation of basal keratinocytes(Xie et al. (1998) Nature 391:90-92).

While not wishing to be bound by any particular theory, the emergingmechanism by which the smo-ptc pathway mediates signal transduction isas follows. In the absence of Hh induction, the activity of Smo isinhibited by Ptc probably through their physical association.Full-length Ci forms a complex with Fu, Cos-2 and suppressor-of-fused[Su(fu)], via which it associates with microtubules. This associationleads to targeting of Ci to the proteasome where it is cleaved torelease the transcriptional repressing form Ci75. The phosphorylation ofCi155 promotes its cleavage, either by promoting association with theCos-2-Fu or by promoting ubiquitination (or both). When Hh binds to Ptc,the inhibitory effect on Smo is suppressed. The resulting activation ofSmo leads to the dissociation of the Fu-Cos-2-Ci complex frommicrotubules. Cleavage of Ci155 is blocked; this or a related form of Cithen presumably enters the nucleus to activate transcription of ptc, gliand other target genes in association with CREB binding protein (CBP).

SUMMARY OF THE INVENTION

One aspect of the present invention makes available methods and reagentsfor inhibiting smoothened-dependent pathway activation. In certainembodiments, the subject methods can be used to counteract thephenotypic effects of unwanted activation of a hedgehog pathway, such asresulting from hedgehog gain-of-function, ptc loss-of-function orsmoothened gain-of-function mutations. For instance, the subject methodcan involve contacting a cell (in vitro or in vivo) with a smoothenedantagonist (defined infra), such as a steroidal alkaloid or other smallmolecule in an amount sufficient to antagonize a smoothened-dependentpathway activation.

Another aspect of the present invention makes available methods andreagents for activating smoothened-dependent pathway activation, e.g, tomimic all or certain of the effects that treatment with a hedgehogprotein might cause. The subject method can involve contacting a cell(in vitro or in vivo) with a smoothened agonist (defined infra) in anamount sufficient to activate a smoothened-dependent pathway.

The subject methods and compounds may be used to regulate proliferationand/or differentiation of cells in vitro and/or in vivo, e.g., in theformation of tissue from stem cells, or to prevent the growth ofhyperproliferative cells to illustrate but a few uses.

The subject compounds may be formulated as a pharmaceutical preparationcomprising a pharmaceutically acceptable excipient. Smoothenedantagonists of the invention and/or preparations comprising them may beadministered to a patient to treat conditions involving unwanted cellproliferation, e.g., cancer and/or tumors (such as medulloblastoma,basal cell carcinoma, etc.), non-malignant hyperproliferative disorders,etc. Smoothened agonists can also be used to regulate the growth anddifferentiation of normal tissues. In certain embodiments, suchcompounds or preparations are administered systemically and/or locally,e.g., topically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents structures of the synthetic compounds AY 9944 andtriparanol, of the plant steriodal alkaloids jervine, cyclopamine, andtomatidine, and of cholesterol.

FIG. 2 relates to a sensitive assay for Shh signaling in NIH-3T3 cells.(A) Purification of cholesterol- and palmitate-modified mouse Sonichedgehog signaling domain ShhN_(p). Detergent-insoluble proteolipidcomplexes were isolated from 293 cells expressing full-length Shh (M. K.Cooper, J. A. Porter, K. E. Young, P. A. Beachy, Science 280, 1603(1998)), and ShhN_(p) was purified to apparent homogeneity byimmunoaffinity chromatography. Although recombinant ShhN lackingcholesterol and palmitate modifications is fully active in neural plateexplant culture assays, this form of ShhN was poorly active in theNIH-3T3 cells. We therefore used the detergent insolubility ofcholesterol-modified ShhN and affinity chromatography to purify theprocessed ShhN protein (ShhN_(p)) from a human 293 cell line engineeredto express the full length mouse Shh construct. Detergent-insolublecomplexes (DIGs) were isolated as described by Brown and Rose (1992),with the following modifications. Cells from a 150 mm dish were lysedand collected in 2 mL of lysis solution (10 mM NaHPO₄, pH 6.5, 150 mMNaCl, 0.5 mM PMSF, 1% Triton X-100, 2 μg/ml Pepstatin A, 10 μg/mlleupeptin, 5 μg/ml aprotinin, 2 μg/ml E64) at 4° C. Eight sucrosedensity steps (35.625-5%, 4.375%/step; made in above solution, withoutdetergent) were layered onto the 40% sucrose lysate and centrifugationproceeded for 2-12 hr. Low-density, flocculent material was collected(from original position of 18.125% step), diluted 5-fold in 10 mMNaHPO₄, pH 6.5, 150 mM NaCl and harvested by centrifugation at 20,000×gfor 15 min (all at 4° C.). Complexes were solubilized at ambienttemperature in 1% n-octyl-α-D-glucopyranoside, 50 mM HEPES, pH 7.5, 150mM NaCl and the single ShhN_(p) immunoaffinity purification stepproceeded essentially as described in Pepinsky et al. (1998), except theanti-Shh-N monoclonal antibody 5E1 was coupled to Affi-Gel 10 (Bio-Rad)at 6.6 mg/ml gel. The mass of the purified species as determined by massspectrometry corresponds to that of the ShhN polypeptide bearingcovalent palmitoyl and cholesteryl adducts: murine ShhN polypeptide,19,574.05 Da; esterified cholesterol, 368.65; palmitoyl mass (in esteror amide linkage), 238.42; sum, 20,181.1. The inset shows samples fromlysate, detergent-insoluble glycolipid complexes (DIGs; 8 lysate sampleequivalents), and purified ShhN_(p) (0.75 μg) as separated in SDS-PAGE(14%) and stained with Coomassie blue. Mass standards migrated asindicated. (B) NIH-3T3 cells respond to ShhN_(p). NIH-3T3 cellscotransfected with Gli-luc reporter and TK promoter-driven Renillaluciferase control were treated with the indicated concentrations ofShhN_(p) for 2 days. Confluent cultures of NIH-3T3 cells were plated at1:6 dilution to 24 or 96-well plates. On the following day, the cellswere transfected with renilla luciferase (pRL-TK or pRL-SV40; Clontech)or β-galactosidase transfection control (10% w/w DNA), Gli-Luc reporter(40%) and the constructs indicated (50%) using Fugene 6 (Roche)transfection reagent (250 ng (24 well plate) or 100 ng (96 well plate)DNA/well, 3:1 ratio (v/w) of reagent to DNA). After the cells hadreached saturation density (1-2 d), they were changed to low serummedium (0.5% bovine calf serum), and treated with the reagents indicatedfor 1-2 d. Firefly and Renilla luciferase and β-galactosidase activitieswere assayed from the cell lysates by luminometry using dual luciferase(Promega) and Galacto-Light (Tropix) kits, respectively. Luciferaseactivities are normalized relative to control; a representativeexperiment is shown. Note that in this and all subsequent reporterassays, TK-Renilla luciferase activity is used as a control fornormalization. (C) Shh pathway activation is sensitive to cyclopamine inNIH-3T3 cells. NIH-3T3 cells transfected as above (in triplicate) weretreated with ShhN_(p) (4 nM) and/or cyclopamine (5 μM) for 2 d asindicated. Normalized luciferase activities are given as fold inductionrelative to control. Error bars indicate one standard deviation. (D) Lowcell density inhibits Shh pathway activity downstream of Smo. Culturesof Shh-LIGHT (open boxes) or SmoA1-LIGHT (filled diamonds) cells wereplated to 96-well plates in a series of twofold dilutions. The NIH-3T3cell clone Shh-LIGHT and Shh-LIGHT2 stably incorporating the Gli-lucreporter and TK-renilla vectors were established by cotransfection witha vector encoding G418 resistance (pSV-Neo), followed by selection withG418 and cell cloning. Subsequently, a clonal subline of Shh-LIGHTexpressing activated Smo (SmoA1-LIGHT) was established using anexpression vector that allows hygromycin selection (pcDNA3.1+ hygro;Invitrogen). The expression of SmoA1 in the cell line was verified byimmunoblotting. The Shh-LIGHT cells were treated with 4 nM ShhN_(p), andGli-luciferase reporter activity was assayed after 24 h. Fold inductionof the reporter (% of maximum is relative to equally dense Shh-LIGHTcontrol culture) and cell densities (% of maximum Renilla luciferaseactivity) were measured at the end of the experiment. Error barsindicate one standard deviation (quadruplicate wells).

FIG. 3 demonstrates how cyclopamine acts by inhibiting the activity ofSmo. (A) Ptc1−/− cells are sensitive to cyclopamine. Fibroblasts fromPtc1−/− embryos were treated with cyclopamine or forskolin as indicated.After 3 d, cells were lysed and β-galactosidase activity relative toprotein concentration was measured. Since β-galactosidase is expressedfrom the Ptc1 locus, its expression reflects the activity of the Shhpathway. A representative experiment is shown. (B) Activated mutants ofSmo are resistant to cyclopamine. Cultures of NIH-3T3 cells weretransfected (in triplicate) with Gli-luciferase reporter, TK-Renillaluciferase control vector and Smo or SmoA1 expression vectors. Smo DNAwas used at 50% w/w, and SmoA1 at 50%, 5%, and 0.5% w/w. Subsequently,the cultures were treated with 5 μM cyclopamine for 2 d. Error barsindicate one standard deviation. The leftmost four bars, shown forcomparison, are as in FIG. 2C. (C) High level expression of Ptc1restores cyclopamine resistant response of SmoA1 to ShhNp. NIH-3T3 cellswere transfected with Gli-luc reporter, TK-Renilla, Ptc1CTD and SmoA1expression vectors (Ptc to Smo DNA ratio=9). Subsequently, the cultureswere treated with ShhNp (2 nM), cyclopamine (5 μM) and/or forskolin (100μM) as indicated for 2 d. Note that SmoA1 activation of pathway isdramatically reduced by high levels of Ptc1 activity (compare to panelB), and that 2 nM ShhNp restores pathway activity even in the presenceof 5 μM cyclopamine. A representative experiment is shown. Tumor-derivedmutant Smo proteins are intrinsically more active than wild type Smo.NIH-3T3 cells were cotransfected with Gli-luc reporter, β-galactosidasetransfection control, and a control vector or an expression vectorencoding the indicated Smo-Renilla luciferase fusion protein. In therepresentative experiment shown, Shh pathway activity and Smo proteinlevels were measured as firefly and Renilla luciferase activitiesrelative to β-galactosidase, respectively. Epitope-tagged Smo andactivated Smo proteins also displayed similar levels of expression inthese cells (not shown).

FIG. 4 depicts a cyclopamine derivative of increased potency. (A)3-Keto, N-aminoethyl aminocaproyl dihydrocinnamoyl cyclopamine (KAADcyclopamine) was synthesized from cyclopamine. Structure of KAADcyclopamine was verified by NMR and mass spectrometry analyses. KAADcyclopamine can block pathway activation by tumor-derived Smo.Shh-LIGHT2 (diamonds) and SmoA1-LIGHT (circles) cells were treated with4 nM ShhN_(p) (Shh-LIGHT2) and increasing concentrations of KAADcyclopamine (both lines) for 2 d. Relative reporter activity isnormalized to maximum. Note the increased inhibitory potency of KAADcyclopamine as compared to cyclopamine in FIG. 3 A-C. (B) KAADcyclopamine can block pathway activation in Ptc1−/− cells. p2^(PTC−/−)cells (these cells are a cloned line derived from Ptc−/− mouse embryonicfibroblasts) were treated with increasing concentrations of cyclopamine(open boxes) or KAAD cyclopamine (filled boxes) for 2 d. The suppressionof pathway activity induced by SmoA1-Renilla by high concentrations ofcyclopamine derivatives did not involve a decrease in the level ofexpression of the Smo construct (not shown). Cells were seeded intoduplicate 96-well plates, allowed to grow to saturation density, andincubated with cyclopamine and KAAD cyclopamine for 2 d. β-galactosidaseactivity was determined using Galacto-Light kit (no inactivation ofendogenous β-gal activity, Tropix). β-galactosidase activities werenormalized to cell mass as determined from a treated duplicate plateusing the Cell Titer 96AQ assay (Promega). Maximum normalizedβ-galactosidase activities (1103 for KAAD-cyclopamine and 916 forcyclopamine) were set to 1 and minimum activities (191 and 144,respectively) were set to 0, Significant toxicity (microscopicallyvisible cell death, or decrease in Cell Titer reading) was not observed,even at the highest doses of compounds used. β-galactosidase activity isnormalized to the maximum. Error bars in A and B indicate one standarddeviation.

FIGS. 5A-5D present inhibitors of the Hedgehog pathway according to thepresent invention.

FIG. 6 depicts response of fibrosarcoma tumors to treatment with asubject compound.

FIG. 7 compares tumor tissue following treatment with tomatidine withtissue treated with a subject compound.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention relates to the discovery that signal transductionpathways regulated by patched (ptc) and/or smoothened can be inhibited,at least in part, by steroidal alkaloids and analogs thereof. As set outin more detail below, we have observed that derivatives of cyclopaminecan inhibit smoothened-dependent activity of the hedgehog pathway. Whilenot wishing to be bound by any particular theory, our data indicatesthat cyclopamine acts at the level of smoothened, directly or indirectlyshifting the steady-state ratio of active and inactive forms ofsmoothened towards the inactive form (e.g., relative to the absence ofthe steroidal alkaloid).

It is, therefore, specifically contemplated that other small molecules,steroidal and non-steroidal in structure, may similarly interfere withaspects of smoothened-mediated signal transduction. For instance, suchcompounds may be useful for inhibiting proliferation and/or inducingdifferentiation of normal tissues (e.g., tissues which express smo orare otherwise hedgehog-responsive). The subject smoothened antagonistsmay also be used to inhibit proliferation (or other biologicalconsequences) in cells or tissues characterized as having a patchedloss-of-function phenotype, a smoothened gain-of-function phenotype or ahedgehog gain-of-function phenotype.

It is also specifically contemplated that, in light of cyclopamine andother small molecules being able to inhibit smoothened-mediated signaltransduction, that activators of smoothened-mediated signal transductioncan be identified, e.g., compounds which directly or indirectly shiftthe steady-state ratio of active and inactive forms of smoothenedtowards the active form. Such compounds may be useful for, toillustrate, inducing proliferation and/or preventing differentiation ofnormal tissues (e.g., tissues which express smo or are otherwisehedgehog-responsive).

In preferred embodiments, the subject inhibitors and activators areorganic molecules having a molecular weight less than 2500 amu, morepreferably less than 1500 amu, and even more preferably less than 750amu, and are capable of inhibiting at least some of the activity of asmoothened signal transduction pathway.

Thus, the methods of the present invention include the use of agents,such as small molecules, which antagonize or activate (as appropriate)smoothened-dependent activity of the hedgehog pathway, resulting in theregulation of repair and/or functional performance of a wide range ofcells, tissues, and organs. For instance, the subject methods havetherapeutic and cosmetic applications ranging from regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liver,pancreas, and other organs arising from the primitive gut, regulation ofhematopoietic function, regulation of skin and hair growth, etc.Moreover, the subject methods can be performed on cells which areprovided in culture (in vitro), or on cells in a whole animal (in vivo).See, for example, PCT publications WO 95/18856 and WO 96/17924 (thespecifications of which are expressly incorporated by reference herein).

In a certain preferred embodiment, the subject smoothened antagonistscan be to treat epithelial cells having a phenotype of ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function employing an agent which antagonizes hedgehog function.For instance, the subject method can be used in treating or preventingbasal cell carcinoma or other hedgehog pathway-related disorders.

In another preferred embodiment, the subject smoothened antagonists andactivators can, as appropriate, be used to modulate proliferation ordifferentiation of pancreatic cells (e.g., ranging from pancreaticprogenitor cells and mature endocrine or exocrine cells), or to regulatethe growth or development of pancreatic tissue, e.g., in vivo or invitro.

In yet another preferred embodiment, the subject method can be used aspart of a treatment regimen for malignant medulloblastoma and otherprimary CNS malignant neuroectodermal tumors.

In another aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, a smoothenedantagonist or activator such as described herein, formulated in anamount sufficient to regulate, in vivo, a smoothened-dependent pathway,e.g., proliferation, differentiation or other biological consequences ofnormal or abnormal function of, for example, ptc, hedgehog orsmoothened.

The subject treatments using the subject compounds can be effective forboth human and animal subjects. Animal subjects to which the inventionis applicable extend to both domestic animals and livestock, raisedeither as pets or for commercial purposes. Examples are dogs, cats,cattle, horses, sheep, hogs, and goats.

II. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The phrase “aberrant modification or mutation” of a gene refers to suchgenetic lesions as, for example, deletions, substitution or addition ofnucleotides to a gene, as well as gross chromosomal rearrangements ofthe gene and/or abnormal methylation of the gene. Likewise,mis-expression of a gene refers to aberrant levels of transcription ofthe gene relative to those levels in a normal cell under similarconditions, as well as non-wild-type splicing of mRNA transcribed fromthe gene.

“Basal cell carcinomas” exist in a variety of clinical and histologicalforms such as nodular-ulcerative, superficial, pigmented, morphealike,fibroepithelioma and nevoid syndrome. Basal cell carcinomas are the mostcommon cutaneous neoplasms found in humans. The majority of new cases ofnonmelanoma skin cancers fall into this category.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

The term “cAMP regulator” refers to an agent which alters the level oractivity of cAMP in a cell, including agents which act upon adenylatecyclase, cAMP phosphodiesterase, or other molecules which, in turn,regulate cAMP levels or activity. Additionally, cAMP regulators, as theterm is used herein, refer to downstream effectors of cAMP activity,such as protein kinase A. “cAMP agonists” refers to that subset of cAMPregulators which increases the level or activity of cAMP in a cell,while “cAMP antagonists” refers to the subset which decreases the levelor activity of cAMP in a cell.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastases. Exemplary carcinomas include: “basal cellcarcinoma”, which is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction;“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Other carcinomatous epithelial growths are “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

“Dental tissue” refers to tissue in the mouth which is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers which canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect.

An “effective amount” of a subject compound, with respect to the subjectmethod of treatment, refers to an amount of the antagonist in apreparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophogeal, epidermal, and hair follicleepithelial cells. Other exemplary epithelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g., tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, “hair” may refer to theaggregate of such hairs. A “hair follicle” refers to one of thetubular-invaginations of the epidermis enclosing the hairs, and fromwhich the hairs grow. “Hair follicle epithelial cells” refers toepithelial cells which surround the dermal papilla in the hair follicle,e.g., stem cells, outer root sheath cells, matrix cells, and inner rootsheath cells. Such cells may be normal non-malignant cells, ortransformed/immortalized cells.

The term “smoothened antagonist” refers to an agent which represses orinduces transcription of target genes, e.g., gli1 and ptc genes, whichin normal cells are induced or repressed by contact of the cell withhedgehog. In addition to altering a smoothened-dependent pathway,preferred smoothened antagonists can be used to overcome a ptcloss-of-function and/or a smoothened gain-of-function. The term“smoothened antagonist” as used herein also refers to any agent that mayact by regulating a downstream effector of the smoothened pathway suchas fused, suppressor of fused, cubitus interruptus, costal-2, etc.,thereby inhibiting smoothened-dependent pathway activation.

The terms “loss-of-function” and “gain-of-function”, as appropriate,refer to an aberrant modification or mutation of, e.g., a ptc gene,hedgehog gene, or smoothened gene, or a decrease or increase in thelevel of expression of such a gene, which results in a phenotype, e.g.,which resembles contacting a cell with a hedgehog protein, such asaberrant activation of a hedgehog pathway or resemble loss of smofunction. The mutation may include a loss of the ability of the ptc orsmo gene product(s) to regulate the level of activity of Ci proteins,e.g., Gli1, Gli2, and Gli3.

As used herein, “immortalized cells” refers to cells which have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

“Internal epithelial tissue” refers to tissue inside the body which hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

The term “LD₅₀” means the dose of a drug which is lethal in 50% of testsubjects.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “prodrug” is intended to encompass compounds which, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties which are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder which alters the skin's regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

As used herein, “transformed cells” refers to cells which havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, wherein R₈ represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In even more preferred embodiments, R₉ and R₁₀ (and optionallyR′₁₀) each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from heteroatoms(preferably 1 to 4), for example, benzene, pyrrole, furan, thiophene,imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF₃, —CN, or the like. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

A “phosphonamidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “phosphoramidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, for example, an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of −Se-alkyl,−Se-alkenyl, −Se-alkynyl, and −Se—(CH₂)_(m)—R₈, m and R₈ being definedabove.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit hedgehogsignaling), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

III. Exemplary Compounds of the Invention

As described in further detail below, it is contemplated that thesubject methods can be carried out using any of a variety of differentsteroidal alkaloids which can be readily identified, e.g., by such drugscreening assays as described herein. Steroidal alkaloids have a fairlycomplex nitrogen-containing nucleus. Two exemplary classes of steroidalalkaloids for use in the subject methods are the Solanum type and theVeratrum type. The above notwithstanding, in a preferred embodiment, themethods and compositions of the present invention make use of compoundshaving a steroidal alkaloid ring system of cyclopamine.

There are more than 50 naturally occurring veratrum alkaloids includingveratramine, cyclopamine, cycloposine, jervine, and muldamine occurringin plants of the Veratrum spp. The Zigadenus spp., death camas, alsoproduces several veratrum-type of steroidal alkaloids includingzygacine. In general, many of the veratrum alkaloids (e.g., jervine,cyclopamine and cycloposine) consist of a modified steroid skeletonattached spiro to a furanopiperidine. A typical veratrum-type alkaloidmay be represented by:

An example of the Solanum type is solanidine. This steroidal alkaloid isthe nucleus (i.e., aglycone) for two important glycoalkaloids, solanineand chaconine, found in potatoes. Other plants in the Solanum familyincluding various nightshades, Jerusalem cherries, and tomatoes alsocontain solanum-type glycoalkaloids. Glycoalkaloids are glycosides ofalkaloids. A typical solanum-type alkaloid may be represented by:

Based on these structures, and the possibility that certain unwantedside effects can be reduced by some manipulation of the structure, awide range of steroidal alkaloids are contemplated as potentialsmoothened antagonists for use in the subject method. For example,compounds useful in the subject methods include steroidal alkaloidsrepresented in the general formulas (I), or unsaturated forms thereofand/or seco-, nor- or homo-derivatives thereof:

wherein, as valence and stability permit,

R₂ and R₃ represent one or more substitutions to the ring to which eachis attached, for each occurrence, independently represent hydrogen,halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl,silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls,phosphonates, phosphines, carbonyls, carboxyls, carboxamides,anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls,selenoethers, ketones, aldehydes, esters, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), carbonate, or —(CH₂)_(m)—R₈;

R₄ and R₅, independently for each occurrence, are absent or representone or more substitutions to the ring to which each is attached,selected from hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls,hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro, thiol, amines,imines, amides, phosphoryls, phosphonates, phosphines, carbonyls,carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers,alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters,sugar, carbamate, carbonate, or —(CH₂)_(m)—R₈;R₆,

R₇, and R′₇, are absent or represent, independently, halogens, alkyls,alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino,nitro, thiol, amines, imines, amides, phosphoryls, phosphonates,phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls,ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers,ketones, aldehydes, esters, or —(CH₂)_(m)—R₈, or

R₆ and R₇, or R₇ and R′₇, taken together form a ring or polycyclic ring,e.g., which is substituted or unsubstituted,

with the proviso that at least one of R₆, R₇, or R′₇ is present andincludes an amine, e.g., as one of the atoms which makes up the ring;

R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or apolycycle; and

m is an integer in the range 0 to 8 inclusive.

In certain embodiments, R₂ represents ═O, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), ester (e.g., attached to the steroid at oxygen),carbonate, or alkoxy. Substituents such as carbamate, ester, carbonate,and alkoxy may be substituted or unsubstituted, e.g., may includeadditional functional groups such as aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc.

In certain embodiments, the amine of R₆, R₇, or R′₇ is a tertiary amine.

In particular embodiments, R₃, for each occurrence, is an —OH, alkyl,—O-alkyl, —C(O)-alkyl, or —C(O)—R₈.

In particular embodiments, R₄, for each occurrence, is an absent, orrepresents —OH, ═O, alkyl, —O-alkyl, —C(O)-alkyl, or —C(O)—R₈.

In particular embodiments, two of R₆, R₇, and R′₇ taken together form anitrogen-containing ring, such as a furanopiperidine, such asperhydrofuro[3,2-b]pyridine, a pyranopiperidine, a quinoline, an indole,a pyranopyrrole, a naphthyridine, a thiofuranopiperidine, or athiopyranopiperidine.

In certain embodiments, the nitrogen-containing ring comprises atertiary amine, e.g., by having an extraannular substitutent on thenitrogen atom, e.g., an alkyl substituted with, for example, aryl,aralkyl, heteroaryl, heteroaralkyl, amide, acylamino, carbonyl, ester,carbamate, urea, ketone, sulfonamide, etc. In certain embodiments, theextraannular substituent of the tertiary amine is a hydrophobicsubstituent. In certain embodiments, the hydrophobic extraannularsubstituent includes an aryl, heteroaryl, carbocyclyl, heterocyclyl, orpolycyclyl group, such as biotin, a zwitterionic complex of boron, asteroidal polycycle, etc. In certain embodiments, the hydrophobicsubstituent may consist essentially of a combination of alkyl, amido,acylamino, ketone, ester, ether, halogen, alkenyl, alkynyl, aryl,aralkyl, urea, or similar functional groups, including between 5 and 40non-hydrogen atoms, more preferably between 5 and 20 non-hydrogen atoms.

In particular embodiments, R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle, or a polycycle, and preferably R₈ is apiperidine, pyrrolidine, pyridine, pyrimidine, morpholine,thiomorpholine, pyridazine, etc.

In certain embodiments, R₂ represents ═O, sugar, carbamate, ester,carbonate, or alkoxy; R₃, for each occurrence, is an —OH, alkyl,—O-alkyl, —C(O)-alkyl, or —C(O)—R₈; R₄, for each occurrence, is absent,or represents —OH, ═O, alkyl, —O-alkyl, —C(O)-alkyl, or —C(O)—R₈; andR₅, for each occurrence, is absent, or represents —OH, ═O, or alkyl.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula Ia orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In certain embodiments, the steroidal alkaloid is represented in thegeneral formula (II), or unsaturated forms thereof and/or seco-, nor- orhomo-derivatives thereof:

wherein R₂, R₃, R₄, R₅, R₆, R₇, and R′₇ are as defined above, and Xrepresents O or S, though preferably O.

In certain embodiments, R₂ represents ═O, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), ester (e.g., attached to the steroid at oxygen),carbonate, or alkoxy. Substituents such as carbamate, ester, carbonate,and alkoxy may be substituted or unsubstituted, e.g., may includeadditional functional groups such as aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc.

In certain embodiments, the amine of R₆, R₇, or R′₇ is a tertiary amine,e.g., substituted with a substituted or unsubstituted alkyl. In certainembodiments, the amine is part of a bicyclic ring system formed from R₇and R′₇, e.g., a furanopiperidine system, and the third substitutent isan alkyl substituted with, for example, aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc. In certain embodiments, the extraannularsubstituent of the tertiary amine is a hydrophobic substituent. Incertain embodiments, the hydrophobic extraannular substituent includesan aryl, heteroaryl, carbocyclyl, heterocyclyl, or polycyclyl group,such as biotin, a zwitterionic complex of boron, a steroidal polycycle,etc. In certain embodiments, the hydrophobic substituent may consistessentially of a combination of alkyl, amido, acylamino, ketone, ester,ether, halogen, alkenyl, alkynyl, aryl, aralkyl, urea, or similarfunctional groups, including between 5 and 40 non-hydrogen atoms, morepreferably between 5 and 20 non-hydrogen atoms.

In certain embodiments, R₂ represents ═O, sugar, carbamate, ester,carbonate, or alkoxy; R₃, for each occurrence, is an —OH, alkyl,—O-alkyl, —C(O)-alkyl, or —C(O)—R₈; R₄, for each occurrence, is absent,or represents —OH, ═O, alkyl, —O-alkyl, —C(O)-alkyl, or —C(O)—R₈; andR₅, for each occurrence, is absent, or represents —OH, ═O, or alkyl.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula IIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In certain embodiments, the steroidal alkaloid is represented in thegeneral formula (III), or unsaturated forms thereof and/or seco-, nor-or homo-derivatives thereof:

wherein

R₂, R₃, R₄, R₅ and R₈ are as defined above;

A and B represent monocyclic or polycyclic groups;

T represents an alkyl, an aminoalkyl, a carboxyl, an ester, an amide,ether or amine linkage of 1-10 bond lengths;

T′ is absent, or represents an alkyl, an aminoalkyl, a carboxyl, anester, an amide, ether or amine linkage of 1-3 bond lengths, wherein ifT and T′ are present together, than T and T′ taken together with thering A or B form a covalently closed ring of 5-8 ring atoms;

R₉ represents one or more substitutions to the ring A or B, which foreach occurrence, independently represent halogens, alkyls, alkenyls,alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro,thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines,carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers,thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones,aldehydes, esters, or —(CH₂)_(m)—R₈; and

n and m are, independently, zero, 1 or 2;

with the proviso that A, or T, T′, and B, taken together, include atleast one amine.

In certain embodiments, R₂ represents ═O, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), ester (e.g., attached to the steroid at oxygen),carbonate, or alkoxy. Substituents such as carbamate, ester, carbonate,and alkoxy may be substituted or unsubstituted, e.g., may includeadditional functional groups such as aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc.

In certain embodiments, the amine of A, or T, T′, and B, is a tertiaryamine, e.g., substituted with a substituted or unsubstituted alkyl,e.g., substituted with aryl, aralkyl, heteroaryl, heteroaralkyl, amide,acylamino, carbonyl, ester, carbamate, urea, ketone, sulfonamide, etc.In certain embodiments, the extraannular substituent of the tertiaryamine is a hydrophobic substituent. In certain embodiments, thehydrophobic extraannular substituent includes an aryl, heteroaryl,carbocyclyl, heterocyclyl, or polycyclyl group, such as biotin, azwitterionic complex of boron, a steroidal polycycle, etc. In certainembodiments, the hydrophobic substituent may consist essentially of acombination of alkyl, amido, acylamino, ketone, ester, ether, halogen,alkenyl, alkynyl, aryl, aralkyl, urea, or similar functional groups,including between 5 and 40 non-hydrogen atoms, more preferably between 5and 20 non-hydrogen atoms.

In certain embodiments, R₂ represents ═O, sugar, carbamate, ester,carbonate, or alkoxy; R₃, for each occurrence, is an —OH, alkyl,—O-alkyl, —C(O)-alkyl, or —C(O)—R₈; R₄, for each occurrence, is absent,or represents —OH, ═O, alkyl, —O-alkyl, —C(O)-alkyl, or —C(O)—R₈; andR₅, for each occurrence, is absent, or represents —OH, ═O, or alkyl.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula IIIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

For example, the subject methods can utilize smoothened antagonistsbased on the veratrum-type steroidal alkaloids jervine, cyclopamine,cycloposine, mukiamine or veratramine, e.g., which may be represented inthe general formula (IV), or unsaturated forms thereof and/or seco-,nor- or homo-derivatives thereof:

wherein

R₂, R₃, R₄, R₅, R₆ and R₉ are as defined above;

R₂₂ is absent or represents an alkyl, an alkoxyl or —OH.

In certain embodiments, R₂ represents ═O, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), ester (e.g., attached to the steroid at oxygen),carbonate, or alkoxy. Substituents such as carbamate, ester, carbonate,and alkoxy may be substituted or unsubstituted, e.g., may includeadditional functional groups such as aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc.

In certain embodiments, R₉ includes a substituent on nitrogen, e.g., asubstituted or unsubstituted alkyl, e.g., substituted with, for example,aryl, aralkyl, heteroaryl, heteroaralkyl, amide, acylamino, carbonyl,ester, carbamate, urea, ketone, sulfonamide, etc. In certainembodiments, the extraannular substituent (e.g., R₉) of the tertiaryamine is a hydrophobic substituent. In certain embodiments, thehydrophobic extraannular substituent includes an aryl, heteroaryl,carbocyclyl, heterocyclyl, or polycyclyl group, such as biotin, azwitterionic complex of boron, a steroidal polycycle, etc. In certainembodiments, the hydrophobic substituent may consist essentially of acombination of alkyl, amido, acylamino, ketone, ester, ether, halogen,alkenyl, alkynyl, aryl, aralkyl, urea, or similar functional groups,including between 5 and 40 non-hydrogen atoms, more preferably between 5and 20 non-hydrogen atoms.

In certain embodiments, R₂ represents ═O, sugar, carbamate, ester,carbonate, or alkoxy; R₃, for each occurrence, is an —OH, alkyl,—O-alkyl, —C(O)-alkyl, or —C(O)—R₈; R₄, for each occurrence, is absent,or represents —OH, ═O, alkyl, —O-alkyl, —C(O)-alkyl, or —C(O)—R₈; andR₅, for each occurrence, is absent, or represents —OH, ═O, or alkyl.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula IVa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In certain embodiments, the steroidal alkaloid is represented in thegeneral formula (V) or unsaturated forms thereof and/or seco-, nor- orhomo-derivatives thereof:

wherein R₂, R₃, R₄, R₆ and R₉ are as defined above;

In certain embodiments, R₂ represents ═O, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), ester (e.g., attached to the steroid at oxygen),carbonate, or alkoxy. Substituents such as carbamate, ester, carbonate,and alkoxy may be substituted or unsubstituted, e.g., may includeadditional functional groups such as aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc.

In certain embodiments, R₉ includes a substituent on nitrogen, e.g., asubstituted or unsubstituted alkyl, e.g., substituted with, for example,aryl, aralkyl, heteroaryl, heteroaralkyl, amide, acylamino, carbonyl,ester, carbamate, urea, ketone, sulfonamide, etc.

In certain embodiments, the extraannular substituent of the tertiaryamine (e.g., R₉) is a hydrophobic substituent. In certain embodiments,the hydrophobic extraannular substituent includes an aryl, heteroaryl,carbocyclyl, heterocyclyl, or polycyclyl group, such as biotin, azwitterionic complex of boron, a steroidal polycycle, etc. In certainembodiments, the hydrophobic substituent may consist essentially of acombination of alkyl, amido, acylamino, ketone, ester, ether, halogen,alkenyl, alkynyl, aryl, aralkyl, urea, or similar functional groups,including between 5 and 40 non-hydrogen atoms, more preferably between 5and 20 non-hydrogen atoms.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula Va orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

Another class of smoothened antagonists can be based on theveratrum-type steroidal alkaloids resmebling verticine and zygacine,e.g., general formula (VI), or unsaturated forms thereof and/or seco-,nor- or homo-derivatives thereof:

-   -   wherein R₂, R₃, R₄, R₅ and R₉ are as defined above;

In certain embodiments, R₂ represents ═O, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), ester (e.g., attached to the steroid at oxygen),carbonate, or alkoxy. Substituents such as carbamate, ester, carbonate,and alkoxy may be substituted or unsubstituted, e.g., may includeadditional functional groups such as aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula VIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In certain embodiments, the steroidal alkaloid is represented in thegeneral formula (VII) or unsaturated forms thereof and/or seco-, nor- orhomo-derivatives thereof:

-   -   wherein R₂, R₃, R₄, R₅ and R₉ are as defined above.

In certain embodiments, R₂ represents ═O, sugar (e.g., monosaccharide,disaccharide, polysaccharide, etc.), carbamate (e.g., attached to thesteroid at oxygen), ester (e.g., attached to the steroid at oxygen),carbonate, or alkoxy. Substituents such as carbamate, ester, carbonate,and alkoxy may be substituted or unsubstituted, e.g., may includeadditional functional groups such as aryl, aralkyl, heteroaryl,heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,ketone, sulfonamide, etc.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula VIIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In certain embodiments, the subject antagonists and activators can bechosen on the basis of their selectively for the smoothened pathway.This selectivity can be for the smoothened pathway versus othersteroid-mediated pathways (such as testosterone or estrogen mediatedactivities), as well as selectivity for particularhedgehog/ptc/smoothened pathways, e.g., which isotype specific for ptc(e.g., ptc-1, ptc-2) or hedgehog (e.g., Shh, Ihh, Dhh, etc). Forinstance, the subject method may employ steroidal alkaloids which do notsubstantially interfere with the biological activity of such steroids asaldosterone, androstane, androstene, androstenedione, androsterone,cholecalciferol, cholestane, cholic acid, corticosterone, cortisol,cortisol acetate, cortisone, cortisone acetate, deoxycorticosterone,digitoxigenin, ergocalciferol, ergosterol, estradiol-17-α,estradiol-17-β, estriol, estrane, estrone, hydrocortisone, lanosterol,lithocholic acid, mestranol, β-methasone, prednisone, pregnane,pregnenolone, progesterone, spironolactone, testosterone, triamcinoloneand their derivatives, at least so far as those activities are unrelatedto ptc related signaling.

In one embodiment, the subject steroidal alkaloid for use in the presentmethod has a k_(d) for members of the nuclear hormone receptorsuperfamily of greater than 1 μM, and more preferably greater than 1 mM,e.g., it does not bind estrogen, testosterone receptors or the like.Preferably, the subject smoothened antagonist has no estrogenic activityat physiological concentrations (e.g., in the range of 1 ng-1 mg/kg).

In this manner, untoward side effects which may be associated certainmembers of the steroidal alkaloid class can be reduced. For example,using the drug screening assays described herein, the application ofcombinatorial and medicinal chemistry techniques to the steroidalalkaloids provides a means for reducing such unwanted negative sideeffects including personality changes, shortened life spans,cardiovascular diseases and vascular occlusion, organ toxicity,hyperglycemia and diabetes, Cushnoid features, “wasting” syndrome,steroidal glaucoma, hypertension, peptic ulcers, and increasedsusceptibility to infections. For certain embodiments, it will bebenefical to reduce the teratogenic activity relative to jervine, as forexample, in the use of the subject method to selectively inhibitspermatogenesis.

In a preferred embodiment, the subject antagonists are steroidalalkaloids other than spirosolane, tomatidine, jervine, etc.

In particular embodiments, the steroidal alkaloid is chosen for usebecause it is more selective for one patched isoform over the next,e.g., 10-fold, and more preferably at least 100- or even 1000-fold moreselective for one patched pathway (ptc-1, ptc-2) over another. Likewise,the steroidal alkaloid may be chosen for use because it is moreselective for one smoothened isoform over the next, e.g., 10-fold, andmore preferably at least 100- or even 1000-fold more selective for onewild-type smoothened protein (should various isoforms exist) or foractivated smoothened mutants relative to wild-type smoothened. Incertain embodiments, the subject method can be carried out conjointlywith the administration of growth and/or trophic factors, orcompositions which also act on other parts of the hedgehog/smoothenedpathway. For instance, it is contemplated that the subject methods caninclude treatment with an agent that modulates cAMP levels, e.g.,increasing or decreasing intracellular levels of cAMP.

In one embodiment, the subject method utilizes a smoothened antagonist,and the conjoint agent elevates cAMP levels in order to enhance theefficacy of the smoothened antagonist.

For example, compounds which may activate adenylate cyclase includeforskolin (FK), cholera toxin (CT), pertussis toxin (PT), prostaglandins(e.g., PGE-1 and PGE-2), colforsin and β-adrenergic receptor agonists.β-Adrenergic receptor agonists (sometimes referred to herein as“β-adrenergic agonists”) include albuterol, bambuterol, bitolterol,carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine,dopexamine, ephedrine, epinephrine, etafedrine, ethylnorepinephrine,fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine,isoproterenol, mabuterol, metaproterenol, methoxyphenamine,norepinephrine, oxyfedrine, pirbuterol, prenalterol, procaterol,propranolol, protokylol, quinterenol, reproterol, rimiterol, ritodrine,salmefamol, soterenol, salmeterol, terbutaline, tretoquinol,tulobuterol, and xamoterol.

Compounds which may inhibit a cAMP phosphodiesterase include aminone,milrinone, xanthine, methylxanthine, anagrelide, cilostamide,medorinone, indolidan, rolipram, 3-isobutyl-1-methylxanthine (IBMX),chelerythrine, cilostazol, glucocorticoids, griseolic acid, etazolate,caffeine, indomethacin, papverine, MDL 12330A, SQ 22536, GDPssS,clonidine, type III and type IV phosphodiesterase inhibitors,methylxanthines such as pentoxifylline, theophylline, theobromine,pyrrolidinones and phenyl cycloalkane and cycloalkene derivatives(described in PCT publications Nos. WO 92/19594 and WO 92/10190),lisophylline, and fenoxamine.

Analogs of cAMP which may be useful in the present method includedibutyryl-cAMP (db-cAMP), (8-(4)-chlorophenylthio)-cAMP (cpt-cAMP),8-[(4-bromo-2,3-dioxobutyl)thio]-cAMP,2-[(4-bromo-2,3-dioxobutyl)thio]-cAMP, 8-bromo-cAMP, dioctanoyl-cAMP,Sp-adenosine 3′:5′-cyclic phosphorothioate, 8-piperidino-cAMP,N⁶-phenyl-cAMP, 8-methylamino-cAMP, 8-(6-aminohexyl)amino-cAMP,2′-deoxy-cAMP, N⁶,2′-O-dibutryl-cAMP, N⁶,2′-O-disuccinyl-cAMP,N⁶-monobutyryl-cAMP, 2′-β-monobutyryl-cAMP,2′-O-monobutryl-8-bromo-cAMP, N⁶-monobutryl-2′-deoxy-cAMP, and2′-O-monosuccinyl-cAMP.

Compounds which may reduce the levels or activity of cAMP includeprostaglandylinositol cyclic phosphate (cyclic PIP), endothelins (ET)-1and -3, norepinepurine, K252a, dideoxyadenosine, dynorphins, melatonin,pertussis toxin, staurosporine, G_(i) agonists, MDL 12330A, SQ 22536,GDPssS and clonidine, beta-blockers, and ligands of G-protein coupledreceptors. Additional compounds are disclosed in U.S. Pat. Nos.5,891,875, 5,260,210, and 5,795,756.

Above-listed compounds useful in the subject methods may be modified toincrease the bioavailability, activity, or other pharmacologicallyrelevant property of the compound. For example, forskolin has theformula:

Modifications of forskolin which have been found to increase thehydrophilic character of forskolin without severely attenuating thedesired biological activity include acylation of the hydroxyls at C6and/or C7 (after removal of the acetyl group) with hydrophilic acylgroups. In compounds wherein C6 is acylated with a hydrophilic acylgroup, C7 may optionally be deacetylated. Suitable hydrophilic acylgroups include groups having the structure —(CO)(CH₂)_(n)X, wherein X isOH or NR₂; R is hydrogen, a C₁-C₄ alkyl group, or two Rs taken togetherform a ring comprising 3-8 atoms, preferably 5-7 atoms, which mayinclude heteroatoms (e.g., piperazine or morpholine rings); and n is aninteger from 1-6, preferably from 1-4, even more preferably from 1-2.Other suitable hydrophilic acyl groups include hydrophilic amino acidsor derivatives thereof, such as aspartic acid, glutamic acid,asparagine, glutamine, serine, threonine, tyrosine, etc., includingamino acids having a heterocyclic side chain. Forskolin, or othercompounds listed above, modified by other possible hydrophilic acyl sidechains known to those of skill in the art may be readily synthesized andtested for activity in the present method.

Similarly, variants or derivatives of any of the above-listed compoundsmay be effective as cAMP antagonists in the subject method, e.g., inorder to decrease cAMP levels and potentiate the activity of asmoothened activator. Those skilled in the art will readily be able tosynthesize and test such derivatives for suitable activity.

IV. Exemplary Applications of Method and Compositions

One aspect of the present invention relates to a method of modulating adifferentiated state, survival, and/or proliferation of a cell, such asa normal cell or a cell having a ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function, by contacting thecells with a compound as set forth above according to the subject methodand as the circumstances may warrant.

For instance, it is contemplated by the invention that, in light of thefindings of an apparently broad involvement of hedgehog, ptc, andsmoothened in the formation of ordered spatial arrangements ofdifferentiated tissues in vertebrates, the subject method could be usedas part of a process for generating and/or maintaining an array ofdifferent vertebrate tissue both in vitro and in vivo. The compound,whether inductive or anti-inductive with respect to proliferation ordifferentiation of a given tissue, can be, as appropriate, any of thepreparations described above.

For example, the present method of using subject compounds is applicableto cell culture techniques wherein it is desirable to control theproliferation or differentiation of the cell. A subject compound may beemployed in a method directed towards cells which have a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype. In vitro neuronal culture systems haveproved to be fundamental and indispensable tools for the study of neuraldevelopment, as well as the identification of neurotrophic factors suchas nerve growth factor (NGF), ciliary trophic factors (CNTF), and brainderived neurotrophic factor (BDNF). One use of the present method may bein cultures of neuronal stem cells, such as in the use of such culturesfor the generation of new neurons and glia. In such embodiments of thesubject method, the cultured cells can be contacted with a compound ofthe present invention in order to alter the rate of proliferation ofneuronal stem cells in the culture and/or alter the rate ofdifferentiation, or to maintain the integrity of a culture of certainterminally differentiated neuronal cells. In an exemplary embodiment,the subject method can be used to culture, for example, sensory neuronsor, alternatively, motorneurons. Such neuronal cultures can be used asconvenient assay systems as well as sources of implantable cells fortherapeutic treatments.

According to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vitro and their rate ofproliferation and/or differentiation can be affected by contact withcompounds of the present invention. Generally, a method is providedcomprising the steps of isolating neural progenitor cells from ananimal, perpetuating these cells in vitro or in vivo, preferably in thepresence of growth factors, and regulating the differentiation of thesecells into particular neural phenotypes, e.g., neurons and glia, bycontacting the cells with a subject compound.

Progenitor cells are thought to be under a tonic inhibitory influencewhich maintains the progenitors in a suppressed state until theirdifferentiation is required. However, recent techniques have beenprovided which permit these cells to be proliferated, and unlike neuronswhich are terminally differentiated and therefore non-dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

By “progenitor” it is meant an oligopotent or multipotent stem cellwhich is able to divide without limit and, under specific conditions,can produce daughter cells which terminally differentiate such as intoneurons and glia. These cells can be used for transplantation into aheterologous or autologous host. By heterologous is meant a host otherthan the animal from which the progenitor cells were originally derived.By autologous is meant the identical host from which the cells wereoriginally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal which contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's Disease patients, or thesubstantia nigra pars compacta which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, in particular during epilepsy surgery, and more particularlyduring temporal lobectomies and hippocampalectomies.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrumentor by mincing with a scalpel to a allow outgrowth of specific cell typesfrom a tissue. Dissociation of fetal cells can be carried out in tissueculture medium, while a preferable medium for dissociation of juvenileand adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSFcontains 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃,and 10 mM D-glucose. Low Ca²⁺ aCSF contains the same ingredients exceptfor MgCl₂ at a concentration of 3.2 mM and CaCl₂ at a concentration of0.1 mM.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12, and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7, even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be control in culture by plating (or resuspending) the cellsin the presence of a subject compound.

To further illustrate other uses of the subject compounds, it is notedthat intracerebral grafting has emerged as an additional approach tocentral nervous system therapies. For example, one approach to repairingdamaged brain tissues involves the transplantation of cells from fetalor neonatal animals into the adult brain (Dunnett et al. (1987) J ExpBiol 123:265-289; and Freund et al. (1985) J Neurosci 5:603-616). Fetalneurons from a variety of brain regions can be successfully incorporatedinto the adult brain, and such grafts can alleviate behavioral defects.For example, movement disorder induced by lesions of dopaminergicprojections to the basal ganglia can be prevented by grafts of embryonicdopaminergic neurons. Complex cognitive functions that are impairedafter lesions of the neocortex can also be partially restored by graftsof embryonic cortical cells. The subject method can be used to regulatethe growth state in the culture, or where fetal tissue is used,especially neuronal stem cells, can be used to regulate the rate ofdifferentiation of the stem cells.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role ofcompounds employed in the present method to culture such stem cells canbe to regulate differentiation of the uncommitted progenitor, or toregulate further restriction of the developmental fate of a committedprogenitor cell towards becoming a terminally differentiated neuronalcell. For example, the present method can be used in vitro to regulatethe differentiation of neural crest cells into glial cells, schwanncells, chromaffin cells, cholinergic sympathetic or parasympatheticneurons, as well as peptidergic and serotonergic neurons. The subjectcompounds can be used alone, or can be used in combination with otherneurotrophic factors which act to more particularly enhance a particulardifferentiation fate of the neuronal progenitor cell.

In addition to the implantation of cells cultured in the presence of thesubject compounds, yet another aspect of the present invention concernsthe therapeutic application of a subject compound to regulate the growthstate of neurons and other neuronal cells in both the central nervoussystem and the peripheral nervous system. The ability of ptc, hedgehog,and smoothened to regulate neuronal differentiation during developmentof the nervous system and also presumably in the adult state indicatesthat, in certain instances, the subject compounds can be expected tofacilitate control of adult neurons with regard to maintenance,functional performance, and aging of normal cells; repair andregeneration processes in chemically or mechanically lesioned cells; andtreatment of degeneration in certain pathological conditions. In lightof this understanding, the present invention specifically contemplatesapplications of the subject method to the treatment protocol of(prevention and/or reduction of the severity of) neurological conditionsderiving from: (i) acute, subacute, or chronic injury to the nervoussystem, including traumatic injury, chemical injury, vascular injury anddeficits (such as the ischemia resulting from stroke), together withinfectious/inflammatory and tumor-induced injury; (ii) aging of thenervous system including Alzheimer's disease; (iii) chronicneurodegenerative diseases of the nervous system, including Parkinson'sdisease, Huntington's chorea, amylotrophic lateral sclerosis, diabeticneuropathy, and the like, as well as spinocerebellar degenerations; and(iv) chronic immunological diseases of the nervous system or affectingthe nervous system, including multiple sclerosis.

As appropriate, the subject method can also be used in generating nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, subject compounds can be added to the prostheticdevice to regulate the rate of growth and regeneration of the dendriticprocesses. Exemplary nerve guidance channels are described in U.S. Pat.Nos. 5,092,871 and 4,955,892.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the subject compounds can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, meningiomas, medulloblastomas,neuroectodermal tumors, and ependymomas.

In a preferred embodiment, the subject method can be used as part of atreatment regimen for malignant medulloblastoma and other primary CNSmalignant neuroectodermal tumors.

In certain embodiments, the subject method is used as part of treatmentprogram for medulloblastoma. Medulloblastoma, a primary brain tumor, isthe most common brain tumor in children. A medulloblastoma is aprimitive neuroectodermal tumor arising in the posterior fossa. Theyaccount for approximately 25% of all pediatric brain tumors (Miller).Histologically, they are small round cell tumors commonly arranged intrue rosettes, but may display some differentiation to astrocytes,ependymal cells or neurons (Rorke; Kleihues). PNET's may arise in otherareas of the brain including the pineal gland (pineoblastoma) andcerebrum. Patients with tumors arising in the supratentorial regiongenerally fare worse than their PF counterparts.

Medulloblastoma/PNET's are known to recur anywhere in the CNS afterresection, and can even metastasize to bone. Pretreatment evaluationshould therefore include an examination of the spinal cord to excludethe possibility of “dropped metastases”. Gadolinium-enhanced MRI haslargely replaced myelography for this purpose, and CSF cytology isobtained postoperatively as a routine procedure.

In other embodiments, the subject method is used as part of treatmentprogram for ependymomas. Ependymomas account for approximately 10% ofthe pediatric brain tumors in children. Grossly, they are tumors thatarise from the ependymal lining of the ventricles and microscopicallyform rosettes, canals, and perivascular rosettes. In the CHOP series of51 children reported with ependymomas, ¾ were histologically benign.Approximately ⅔ arose from the region of the 4th ventricle. One thirdpresented in the supratentorial region. Age at presentation peaksbetween birth and 4 years, as demonstrated by SEER data as well as datafrom CHOP. The median age is about 5 years. Because so many childrenwith this disease are babies, they often require multimodal therapy.

Yet another aspect of the present invention concerns the observation inthe art that ptc, hedgehog, and/or smoothened are involved inmorphogenic signals involved in other vertebrate organogenic pathways inaddition to neuronal differentiation as described above, having apparentroles in other endodermal patterning, as well as both mesodermal andendodermal differentiation processes. Thus, it is contemplated by theinvention that compositions comprising one or more of the subjectcompounds can also be utilized for both cell culture and therapeuticmethods involving generation and maintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thatptc, hedgehog, and smoothened are apparently involved in controlling thedevelopment of stem cells responsible for formation of the digestivetract, liver, lungs, and other organs which derive from the primitivegut. Shh serves as an inductive signal from the endoderm to themesoderm, which is critical to gut morphogenesis. Therefore, forexample, compounds of the instant method can be employed for regulatingthe development and maintenance of an artificial liver which can havemultiple metabolic functions of a normal liver. In an exemplaryembodiment, the subject method can be used to regulate the proliferationand differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, therapeutic compositions of subject compounds canbe utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisingsubject compounds can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4. The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homeobox 1), an essential regulator of earlypancreatic development. Apelqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specialisedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1− Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Cajal, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject compounds can be used to control or regulate theproliferation and/or differentiation of pancreatic tissue both in vivoand in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the inhibitors of the presentinvention may provide therapeutic benefits, with the general strategybeing, for example, the correction of aberrant insulin expression, ormodulation of differentiation. More generally, however, the presentinvention relates to a method of inducing and/or maintaining adifferentiated state, enhancing survival and/or affecting proliferationof pancreatic cells, by contacting the cells with the subjectinhibitors. For instance, it is contemplated by the invention that, inlight of the apparent involvement of ptc, hedgehog, and smoothened inthe formation of ordered spatial arrangements of pancreatic tissues, thesubject method could be used as part of a technique to generate and/ormaintain such tissue both in vitro and in vivo. For instance, modulationof the function of hedgehog can be employed in both cell culture andtherapeutic methods involving generation and maintenance β-cells andpossibly also for non-pancreatic tissue, such as in controlling thedevelopment and maintenance of tissue from the digestive tract, spleen,lungs, urogenital organs (e.g., bladder), and other organs which derivefrom the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders affecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells which can result inalterations of insulin secretory capacity of the pancreas. For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of β-cells or decreasedislet cell mass. To the extent that aberrant ptc, hedgehog, andsmoothened signaling may be indicated in disease progression, thesubject regulators can be used to enhance regeneration of the tissueafter anti-tumor therapy.

Moreover, manipulation of hedgehog signaling properties at differentpoints may be useful as part of a strategy for reshaping/repairingpancreatic tissue both in vivo and in vitro. In one embodiment, thepresent invention makes use of the apparent involvement of ptc,hedgehog, and smoothened in regulating the development of pancreatictissue. In general, the subject method can be employed therapeuticallyto regulate the pancreas after physical, chemical or pathologicalinsult. In yet another embodiment, the subject method can be applied tocell culture techniques, and in particular, may be employed to enhancethe initial generation of prosthetic pancreatic tissue devices.Manipulation of proliferation and differentiation of pancreatic tissue,for example, by altering hedgehog activity, can provide a means for morecarefully controlling the characteristics of a cultured tissue. In anexemplary embodiment, the subject method can be used to augmentproduction of prosthetic devices which require β-islet cells, such asmay be used in the encapsulation devices described in, for example, theAebischer et al. U.S. Pat. No. 4,892,538, the Aebischer et al. U.S. Pat.No. 5,106,627, the Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat.No. 4,353,888. Early progenitor cells to the pancreatic islets aremultipotential, and apparently coactivate all the islet-specific genesfrom the time they first appear. As development proceeds, expression ofislet-specific hormones, such as insulin, becomes restricted to thepattern of expression characteristic of mature islet cells. Thephenotype of mature islet cells, however, is not stable in culture, asreappearance of embryonal traits in mature β-cells can be observed. Byutilizing the subject compounds, the differentiation path orproliferative index of the cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of hedgehog function to affect tissue differentiation canbe utilized as a means of maintaining graft viability.

Bellusci et al. (1997) Development 124:53 report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema.

Fujita et al. (1997) Biochem Biophys Res Commun 238:658 reported thatSonic hedgehog is expressed in human lung squamous carcinoma andadenocarcinoma cells. The expression of Sonic hedgehog was also detectedin the human lung squamous carcinoma tissues, but not in the normal lungtissue of the same patient. They also observed that Sonic hedgehogstimulates the incorporation of BrdU into the carcinoma cells andstimulates their cell growth, while anti-Shh-N inhibited their cellgrowth. These results suggest that a ptc, hedgehog, and/or smoothened isinvolved in the cell growth of such transformed lung tissue andtherefore indicates that the subject method can be used as part of atreatment of lung carcinoma and adenocarcinomas, and other proliferativedisorders involving the lung epithelia.

Many other tumors may, based on evidence such as involvement of thehedgehog pathway in these tumors, or detected expression of hedgehog orits receptor in these tissues during development, be affected bytreatment with the subject compounds. Such tumors include, but are by nomeans limited to, tumors related to Gorlin's syndrome (e.g., basal cellcarcinoma, medulloblastoma, meningioma, etc.), tumors evidenced in pctknock-out mice (e.g., hemangioma, rhabdomyosarcoma, etc.), tumorsresulting from gli-1 amplification (e.g., glioblastoma, sarcoma, etc.),tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma,thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone cancer,etc.), Shh-induced tumors (e.g., lung cancer, chondrosarcomas, etc.),and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidney,bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinalcancer (e.g., stomach, intestine, etc.), etc.).

In still another embodiment of the present invention, compositionscomprising one or more of the subject compounds can be used in the invitro generation of skeletal tissue, such as from skeletogenic stemcells, as well as the in vivo treatment of skeletal tissue deficiencies.The present invention particularly contemplates the use of subjectcompounds to regulate the rate of chondrogenesis and/or osteogenesis. By“skeletal tissue deficiency”, it is meant a deficiency in bone or otherskeletal connective tissue at any site where it is desired to restorethe bone or connective tissue, no matter how the deficiency originated,e.g. whether as a result of surgical intervention, removal of tumor,ulceration, implant, fracture, or other traumatic or degenerativeconditions.

For instance, the method of the present invention can be used as part ofa regimen for restoring cartilage function to a connective tissue. Suchmethods are useful in, for example, the repair of defects or lesions incartilage tissue which is the result of degenerative wear such as thatwhich results in arthritis, as well as other mechanical derangementswhich may be caused by trauma to the tissue, such as a displacement oftorn meniscus tissue, meniscectomy, a laxation of a joint by a tornligament, malignment of joints, bone fracture, or by hereditary disease.The present reparative method is also useful for remodeling cartilagematrix, such as in plastic or reconstructive surgery, as well asperiodontal surgery. The present method may also be applied to improvinga previous reparative procedure, for example, following surgical repairof a meniscus, ligament, or cartilage. Furthermore, it may prevent theonset or exacerbation of degenerative disease if applied early enoughafter trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of a subject compound to regulate a cartilage repairresponse in the connective tissue by managing the rate ofdifferentiation and/or proliferation of chondrocytes embedded in thetissue. Such connective tissues as articular cartilage, interarticularcartilage (menisci), costal cartilage (connecting the true ribs and thesternum), ligaments, and tendons are particularly amenable to treatmentin reconstructive and/or regenerative therapies using the subjectmethod. As used herein, regenerative therapies include treatment ofdegenerative states which have progressed to the point of whichimpairment of the tissue is obviously manifest, as well as preventivetreatments of tissue where degeneration is in its earliest stages orimminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g., a sports injury orexcessive wear) or osteoarthritis. The subject regulators may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In some instances, the injected agent can be in theform of a hydrogel or other slow release vehicle in order to permit amore extended and regular contact of the agent with the treated tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue: such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B:74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers which degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own ear or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted witha subject compound during certain stages of the culturing process inorder to manage the rate of differentiation of chondrocytes and theformation of hypertrophic chrondrocytes in the culture.

In another embodiment, the implanted device is treated with a subjectcompound in order to actively remodel the implanted matrix and to makeit more suitable for its intended function. As set out above withrespect to tissue transplants, the artificial transplants suffer fromthe same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The ability to regulate the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as partof a regimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient. Indian hedgehog isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of acompound of the present invention can be employed as part of a methodfor regulating the rate of bone loss in a subject. For example,preparations comprising subject compounds can be employed, for example,to control endochondral ossification in the formation of a “model” forossification.

In yet another embodiment of the present invention, a subject compoundcan be used to regulate spermatogenesis. The hedgehog proteins,particularly Dhh, have been shown to be involved in the differentiationand/or proliferation and maintenance of testicular germ cells. Dhhexpression is initiated in Sertoli cell precursors shortly after theactivation of Sry (testicular determining gene) and persists in thetestis into the adult. Males are viable but infertile, owing to acomplete absence of mature sperm. Examination of the developing testisin different genetic backgrounds suggests that Dhh regulates both earlyand late stages of spermatogenesis. Bitgood et al. (1996) Curr Biol6:298. In a preferred embodiment, the subject compound can be used as acontraceptive. In similar fashion, compounds of the subject method arepotentially useful for modulating normal ovarian function.

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a subject compoundeffective to alter the growth state of a treated epithelial tissue. Themode of administration and dosage regimens will vary depending on theepithelial tissue(s) which is to be treated. For example, topicalformulations will be preferred where the treated tissue is epidermaltissue, such as dermal or mucosal tissues.

A method which “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts) when used together with themethod of the present invention.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g., resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, disposethe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating factors. According to the present invention, atreatment for such ulcers which includes application of a subjectcompound can reduce the abnormal proliferation and differentiation ofthe affected epithelium, helping to reduce the severity of subsequentinflammatory events.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

In other embodiments, antiproliferative preparations of subjectcompounds can be used to inhibit lens epithelial cell proliferation toprevent post-operative complications of extracapsular cataractextraction. Cataract is an intractable eye disease and various studieson a treatment of cataract have been made. But at present, the treatmentof cataract is attained by surgical operations. Cataract surgery hasbeen applied for a long time and various operative methods have beenexamined. Extracapsular lens extraction has become the method of choicefor removing cataracts. The major medical advantages of this techniqueover intracapsular extraction are lower incidence of aphakic cystoidmacular edema and retinal detachment. Extracapsular extraction is alsorequired for implantation of posterior chamber type intraocular lenseswhich are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the highincidence of posterior lens capsule opacification, often calledafter-cataract, which can occur in up to 50% of cases within three yearsafter surgery. After-cataract is caused by proliferation of equatorialand anterior capsule lens epithelial cells which remain afterextracapsular lens extraction. These cells proliferate to causeSommerling rings, and along with fibroblasts which also deposit andoccur on the posterior capsule, cause opacification of the posteriorcapsule, which interferes with vision. Prevention of after-cataractwould be preferable to treatment. To inhibit secondary cataractformation, the subject method provides a means for inhibitingproliferation of the remaining lens epithelial cells. For example, suchcells can be induced to remain quiescent by instilling a solutioncontaining a preparation of a subject compound into the anterior chamberof the eye after lens removal. Furthermore, the solution can beosmotically balanced to provide minimal effective dosage when instilledinto the anterior chamber of the eye, thereby inhibiting subcapsularepithelial growth with some specificity.

The subject method can also be used in the treatment of corneopathiesmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial downgrowth or squamouscell carcinomas of the ocular surface.

Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins canregulate mitogenesis and photoreceptor differentiation in the vertebrateretina, and Ihh is a candidate factor from the pigmented epithelium topromote retinal progenitor proliferation and photoreceptordifferentiation. Likewise, Jensen et al. (1997) Development 124:363demonstrated that treatment of cultures of perinatal mouse retinal cellswith the amino-terminal fragment of Sonic hedgehog results in anincrease in the proportion of cells that incorporate bromodeoxuridine,in total cell numbers, and in rod photoreceptors, amacrine cells andMuller glial cells, suggesting that Sonic hedgehog promotes theproliferation of retinal precursor cells. Thus, the subject method canbe used in the treatment of proliferative diseases of retinal cells andregulate photoreceptor differentiation.

Yet another aspect of the present invention relates to the use of thesubject method to control hair growth. Hair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cystine. Each individual hair comprises a cylindricalshaft and a root, and is contained in a follicle, a flask-likedepression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment. The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

In certain embodiments, the subject method can be employed as a way ofreducing the growth of human hair as opposed to its conventional removalby cutting, shaving, or depilation. For instance, the present method canbe used in the treatment of trichosis characterized by abnormally rapidor dense growth of hair, e.g. hypertrichosis. In an exemplaryembodiment, subject compounds can be used to manage hirsutism, adisorder marked by abnormal hairiness. The subject method can alsoprovide a process for extending the duration of depilation.

Moreover, because a subject compound will often be cytostatic toepithelial cells, rather than cytotoxic, such agents can be used toprotect hair follicle cells from cytotoxic agents which requireprogression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, subject compounds can be used for patientsundergoing chemo- or radiation-therapies which ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death which mightotherwise result from activation of cell death programs. After thetherapy has concluded, the instant method can also be removed withconcommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic preparation of a subjectcompound can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention, the subject method can be used toinduce differentiation and/or inhibit proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a subjectcompound, e.g., which promotes quiescense or differentiation, can beused to treat varying forms of psoriasis, be they cutaneous, mucosal orungual. Psoriasis, as described above, is typically characterized byepidermal keratinocytes which display marked proliferative activationand differentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject method. Actinic keratoses, for example, are superficialinflammatory premalignant tumors arising on sun-exposed and irradiatedskin. The lesions are erythematous to brown with variable scaling.Current therapies include excisional and cryosurgery. These treatmentsare painful, however, and often produce cosmetically unacceptablescarring. Accordingly, treatment of keratosis, such as actinickeratosis, can include application, preferably topical, of a subjectcompound composition in amounts sufficient to inhibit hyperproliferationof epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated bythe subject method. Acne vulgaris, for instance, is a multifactorialdisease most commonly occurring in teenagers and young adults, and ischaracterized by the appearance of inflammatory and noninflammatorylesions on the face and upper trunk. The basic defect which gives riseto acne vulgaris is hypercornification of the duct of a hyperactivesebaceous gland. Hypercornification blocks the normal mobility of skinand follicle microorganisms, and in so doing, stimulates the release oflipases by Propinobacterium acnes and Staphylococcus epidermidisbacteria and Pitrosporum ovale, a yeast. Treatment with anantiproliferative subject compound, particularly topical preparations,may be useful for preventing the transitional features of the ducts,e.g. hypercornification, which lead to lesion formation. The subjecttreatment may further include, for example, antibiotics, retinoids andantiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions which are either pruritic, erythematous, scaly,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales. The subject method can also be used in the treatment ofstasis dermatitis, an often chronic, usually eczematous dermatitis.Actinic dermatitis is dermatitis that due to exposure to actinicradiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subject methodcan be used in the treatment and/or prevention of certain symptoms ofdermatitis caused by unwanted proliferation of epithelial cells. Suchtherapies for these various forms of dermatitis can also include topicaland systemic corticosteroids, antipuritics, and antibiotics.

Ailments which may be treated by the subject method are disordersspecific to non-humans, such as mange.

In still another embodiment, the subject method can be used in thetreatment of human cancers, particularly basal cell carcinomas and othertumors of epithelial tissues such as the skin. For example, subjectcompounds can be employed, in the subject method, as part of a treatmentfor basal cell nevus syndrome (BCNS), and other other human carcinomas,adenocarcinomas, sarcomas and the like.

In a preferred embodiment, the subject method is used as part of atreatment of prophylaxis regimen for treating (or preventing) basal cellcarcinoma. The deregulation of the hedgehog signaling pathway may be ageneral feature of basal cell carcinomas caused by ptc mutations.Consistent overexpression of human ptc mRNA has been described in tumorsof familial and sporadic BCCs, determined by in situ hybridization.Mutations that inactivate ptc may be expected to result inoverexpression of mutant Ptc, because ptc displays negativeautoregulation. Prior research demonstrates that overexpression ofhedgehog proteins can also lead to tumorigenesis. That sonic hedgehog(Shh) has a role in tumorigenesis in the mouse has been suggested byresearch in which transgenic mice overexpressing Shh in the skindeveloped features of BCNS, including multiple BCC-like epidermalproliferations over the entire skin surface, after only a few days ofskin development. A mutation in the Shh human gene from a BCC was alsodescribed; it was suggested that Shh or other Hh genes in humans couldact as dominant oncogenes in humans. Sporadic ptc mutations have alsobeen observed in BCCs from otherwise normal individuals, some of whichare UV-signature mutations. In one recent study of sporadic BCCs, fiveUV-signature type mutations, either CT or CCTT changes, were found outof fifteen tumors determined to contain ptc mutations. Another recentanalysis of sporadic ptc mutations in BCCs and neuroectodermal tumorsrevealed one CT change in one of three ptc mutations found in the BCCs.See, for example, Goodrich et al. (1997) Science 277:1109-13; Xie et al.(1997) Cancer Res 57:2369-72; Oro et al. (1997) Science 276:817-21; Xieet al. (1997) Genes Chromosomes Cancer 18:305-9; Stone et al. (1996)Nature 384:129-34; and Johnson et al. (1996) Science 272:1668-71.

The subject method can also be used to treat patients with BCNS, e.g.,to prevent BCC or other effects of the disease which may be the resultof ptc loss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function. Basal cell nevus syndrome is a rare autosomal dominantdisorder characterized by multiple BCCs that appear at a young age. BCNSpatients are very susceptible to the development of these tumors; in thesecond decade of life, large numbers appear, mainly on sun-exposed areasof the skin. This disease also causes a number of developmentalabnormalities, including rib, head and face alterations, and sometimespolydactyl), syndactyl), and spina bifida. They also develop a number oftumor types in addition to BCCs: fibromas of the ovaries and heart,cysts of the skin and jaws, and in the central nervous system,medulloblastomas and meningiomas. The subject method can be used toprevent or treat such tumor types in BCNS and non-BCNS patients. Studiesof BCNS patients show that they have both genomic and sporadic mutationsin the ptc gene, suggesting that these mutations are the ultimate causeof this disease.

In another aspect, the present invention provides pharmaceuticalpreparations and methods for controlling the formation ofmegakaryocyte-derived cells and/or controlling the functionalperformance of megakaryocyte-derived cells. For instance, certain of thecompositions disclosed herein may be applied to the treatment orprevention of a variety hyperplastic or neoplastic conditions affectingplatelets.

In another aspect, the present invention provides pharmaceuticalpreparations comprising the subject compounds. The compounds for use inthe subject method may be conveniently formulated for administrationwith a biologically acceptable and/or sterile medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thesubject compounds, its use in the pharmaceutical preparation of theinvention is contemplated. Suitable vehicles and their formulationinclusive of other proteins are described, for example, in the bookRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences. Mack Publishing Company, Easton, Pa., USA 1985). Thesevehicles include injectable “deposit formulations”.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of thesubject compounds suitable for veterinary uses, e.g., for the treatmentof live stock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a subject compound at a particulartarget site.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, controlled release patch, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral and topicaladministrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable and/or sterile carriers andcan also be administered in conjunction with other antimicrobial agentssuch as penicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

V. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The subject compoundsaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine. In certainembodiments, the compound included in the pharmaceutical preparation maybe active itself, or may be a prodrug, e.g., capable of being convertedto an active compound in a physiological setting.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water. In certain embodiments,the pharmaceutical preparation is non-pyrogenic, i.e., does not elevatethe body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect, e.g., by overcoming a ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function, in at least asub-population of cells in an animal and thereby blocking the biologicalconsequences of that pathway in the treated cells, at a reasonablebenefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject regulatorsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

It is known that sterols, such as cholesterol, will form complexes withcyclodextrins. Thus, in preferred embodiments, where the inhibitor is asteroidal alkaloid, it may be formulated with cyclodextrins, such as α-,β- and γ-cyclodextrin, dimethyl-β cyclodextrin and2-hydroxypropyl-β-cyclodextrin.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the subject compounds inthe proper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” 0 and B books, Corvallis, Ore., U.S.A., 1977).

VI. Synthetic Schemes and Identification of Active Regulators

The subject compounds, and derivatives thereof, can be prepared readilyby employing known synthetic methodology. As is well known in the art,these coupling reactions are carried out under relatively mildconditions and tolerate a wide range of “spectator” functionality.Additional compounds may be synthesized and tested in a combinatorialfashion, to facilitate the identification of additional compounds whichmay be employed in the subject method.

a. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g. a variegated library of compounds representedabove, can be screened rapidly in high throughput assays in order toidentify potential hedgehog regulator lead compounds, as well as torefine the specificity, toxicity, and/or cytotoxic-kinetic profile of alead compound. For instance, ptc, hedgehog, or smoothened bioactivityassays, such as may be developed using cells with either a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function, can be used to screen a library of the subjectcompounds for those having agonist activity toward ptc or antagonistactivity towards hedgehog or smoothened. Alternatively, bioactivityassays using cells with either a ptc gain-of-function, hedgehogloss-of-function, or smoothened loss-of-function, can be used to screena library of the subject compounds for those having antagonist activitytoward ptc or agonist activity towards hedgehog or smoothened.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectcompounds. See, for example, Blondelle et al. (1995) Trends Anal. Chem.14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899: the EllmanU.S. Pat. No. 5,288,514: the Still et al. PCT publication WO 94/08051;the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen et al. (1994)JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publicationsWO92/10092, WO93/09668 and WO91/07087; and the Lerner et al. PCTpublication WO93/20242). Accordingly, a variety of libraries on theorder of about 100 to 1,000,000 or more diversomers of the subjectcompounds can be synthesized and screened for particular activity orproperty.

In an exemplary embodiment, a library of candidate compound diversomerscan be synthesized utilizing a scheme adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group,optionally located at one of the positions of the candidate regulatorsor a substituent of a synthetic intermediate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. The bead library can then be “plated” with, for example, ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function cells for which a smoothened antagonist is sought. Thediversomers can be released from the bead, e.g. by hydrolysis.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds which may be tested asregulators of hedgehog function.

b. Screening Assays

There are a variety of assays available for determining the ability of acompound such as a hedgehog regulator to regulate ptc, smoothened, orhedgehog function, many of which can be disposed in high-throughputformats. In many drug screening programs which test libraries ofcompounds and natural extracts, high throughput assays are desirable inorder to maximize the number of compounds surveyed in a given period oftime. Thus, libraries of synthetic and natural products can be sampledfor other compounds which are hedgehog regulators.

In addition to cell-free assays, test compounds can also be tested incell-based assays. In one embodiment, cells which have a ptcloss-of-function, hedgehog gain-of-function, or smoothenedgain-of-function phenotype can be contacted with a test agent ofinterest, with the assay scoring for, e.g., inhibition of proliferationof the cell in the presence of the test agent.

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, transcription factors of thecubitus interruptus (ci) family, the serine/threonine kinase fused (fu)and the gene products of costal-2, smoothened and suppressor of fused.

The induction of cells by hedgehog proteins sets in motion a cascadeinvolving the activation and inhibition of downstream effectors, theultimate consequence of which is, in some instances, a detectable changein the transcription or translation of a gene. Potential transcriptionaltargets of hedgehog-mediated signaling are the patched gene (Hidalgo andIngham, 1990 Development 110, 291-301; Marigo et al., 1996) and thevertebrate homologs of the drosophila cubitus interruptus gene, the GLIgenes (Hui et al. (1994) Dev Biol 162:402-413). Patched gene expressionhas been shown to be induced in cells of the limb bud and the neuralplate that are responsive to Shh. (Marigo et al. (1996) PNAS 93:9346-51;Marigo et al. (1996) Development 122:1225-1233). The Gli genes encodeputative transcription factors having zinc finger DNA binding domains(Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al. (1990) MolCell Biol 10:634-642). Transcription of the Gli gene has been reportedto be upregulated in response to hedgehog in limb buds, whiletranscription of the Gli3 gene is downregulated in response to hedgehoginduction (Marigo et al. (1996) Development 122:1225-1233). By selectingtranscriptional regulatory sequences from such target genes, e.g., frompatched or Gli genes, that are responsible for the up- ordown-regulation of these genes in response to hedgehog signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify hedgehog-mediated signalling pathways.Expression of the reporter gene, thus, provides a valuable screeningtool for the development of compounds that act as regulators ofhedgehog.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc loss-of-function, hedgehoggain-of-function, smoothened gain-of-function, or stimulation by Shhitself. The amount of transcription from the reporter gene may bemeasured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic biological activity. The amount of expression from thereporter gene is then compared to the amount of expression in either thesame cell in the absence of the test compound or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdecrease in the amount of transcription indicates that the test compoundhas in some manner agonized the normal ptc signal (or antagonized thegain-of-function hedgehog or smoothened signal), e.g., the test compoundis a potential smoothened antagonist.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Steroidal Compounds

Hedgehog signaling pathways are required for normal embryonicdevelopment yet also are associated with carcinogenesis (L. V. Goodrichand M. P. Scott, Neuron 21, 1243 (1998)). Loss of Sonic hedgehogsignaling (Shh), for example, can result in cyclopia and otherdevelopmental defects of the face, forebrain, and other organs andstructures (C. Chiang et al., Nature 383, 407 (1996)), whereasinappropriate activation of the pathway is associated with basal cellcarcinoma, medulloblastoma, and other neoplastic disorders (H. Hahn etal., Cell 85, 841 (1996); R. L. Johnson et al., Science 272, 1668(1996); M. Gailani et al., Nat Genet. 14, 78 (1996); T. Pietsch et al.,Cancer Res 57, 2085 (1997); J. Reifenberger et al., Cancer Res. 58, 1798(1998); C. Raffel et al., Cancer Res 57, 842 (1997); J. Xie et al.,Nature 391, 90 (1998)). Pharmacological manipulation of this pathwaythus might help elucidate the mechanisms of signal transduction and alsoprovide a practical means to prevent or remedy somatic and congenitalabnormalities. Cyclopamine, a plant steroidal alkaloid, has long beenknown to induce cyclopia and other manifestations of severe HPE invertebrate embryos (R. F. Keeler and W. Binns, Teratology 1, 5 (1968))and more recently was shown to act by inhibiting the cellular responseto the Shh signal (M. K. Cooper, J. A. Porter, K. E. Young, P. A.Beachy, Science 280, 1603 (1998); J. P. Incardona, W. Gaffield, R. P.Kapur, H. Roelink, Development 125, 3553 (1998)). To evaluate thetherapeutic potential of cyclopamine, we investigated the mechanism bywhich it acts.

Cellular responses to the Hh signal are controlled by two multi-passtransmembrane proteins, Smo and Ptc1, predicted to have seven and twelvetransmembrane spans, respectively. Smo is related to the Frizzled familyof Wnt receptors and more distantly to the secretin family of Gprotein-coupled receptors (M. R. Barnes, D. M. Duckworth, L. J. Beeley,Trends Pharmacol Sci 19, 399 (1998)). Genetic and biochemical evidencesuggests that Ptc suppresses the activity of Smo, and that Hh binding toPtc relieves this suppression, allowing activation of downstream targetsthrough the Ci/GLI family of transcriptional effectors (P. Aza-Blanc,F.-A. Remírez-Weber, M.-P. Laget, C. Schwartz, T. B. Kornberg, Cell 89,1043 (1997); N. Methot and K. Basler, Cell 96, 819 (1999); C. H. Chen etal., Cell 98, 305 (1999); B. Wang, J. Fallon, P. Beachy, Cell 100, 423(2000)). To establish a cultured cell based assay that is sensitive tocyclopamine (a previously established Hedgehog signaling assay inDrosophila c1-8 cells (Chen et al., 1999) is resistant to cyclopamine(not shown)), we screened several vertebrate cell lines for atranscriptional response to fully modified ShhN_(p) (FIG. 2A) using aluciferase reporter driven by a promoter comprising eight synthetic Glibinding sites fused to the lens crystallin minimal promoter (H. Sasaki,C.-C. Hui, M. Nakafuku, H. Kondoh, Development 124, 1313 (1997)). Amongseveral fibroblast cell lines that respond to ShhN_(p), NIH-3T3 mouseembryonic fibroblasts, which respond with a 20-150 fold induction ofluciferase activity (FIG. 2B), were selected for further studies.Importantly, when the cells were treated with cyclopamine, thisinduction by ShhN_(p) was completely abolished (FIG. 2C).

Similar to the Hh signaling assay in Drosophila c1-8 cells, the responseto induction by ShhN_(p) required functional Gli binding sites in thereporter (not shown), and the response was augmented by overexpressionof Smo and suppressed by overexpression of Ptc or of activated PKA(Table 1). Pharmacological activation of endogenous PKA by forskolinalso prevented induction of reporter expression (Table 1). Using thisassay, we confirmed the results of Xie et al. that the tumor mutationW539L (SmoA1) constitutively activates Smo, and found that anothermutation from tumor tissue, S537N (SmoA2) also activates Smo. Expressionof either of the activating mutants induced reporter expression to alevel comparable to that observed in ShhN_(p)-treated cells. To furtherexamine the validity of this signaling assay we transfected cells withconstructs encoding known pathway components or treated cells with knowninhibitors of the pathway and analyzed the effects of these treatments,alone and in combination, on reporter activation (Table 1). The majorfindings of Drosophila and mouse genetic analyses were confirmed,indicating that NIH-3T3 cells provide a faithful and physiologicallymeaningful model for analysis of the Shh signaling pathway.

TABLE 1 Luciferase activity from a Gli-dependent reporter as induced bycombinations of Shh pathway inducing and suppressing treatmentsSuppressor Inducer None Ptc cyclopamine forskolin PKA Gli3-N None − − −− − − ShhN_(p) +++ + − − − − Smo + − − − − ND activated Smo +++ +* +* −− − Gli2 ++ ++ ++ ++ + − *Higher than normal dose required to completelysuppress Gli3-N = Gli3 truncated at residue 700, generating a repressorform Expression constructs used: pRK5 for full length mouse Ptc1,C-terminally truncated Ptc1-CTD, Gli3 (1-700) repressor and active PKAcDNAs, and pGE (transient transfections) or pcDNA3.1 + hygro (stablelines; Invitrogen) for the various Smo cDNAs. The CMV promoter drivenmammalian expression vector pGE was derived from pEGFP-C1 (Clontech) byremoving the sequences encoding the EGFP. Renilla luciferase (pRL-TK)was fused to the C-terminus of the Smo open reading frame. The resultingfusion protein constructs had comparable activity to correspondinguntagged constructs in the NIH-3T3 assay

We then made several clonal NIH-3T3 cell lines that contain stablyintegrated reporter. In the best-responding cell lines, we observed20-60 fold induction of luciferase activity by ShhN_(p). Using thesecell lines, we found that a full response to ShhN_(p) required thatcells have reached saturation density, with a reduction in responseobserved for less dense cultures (FIG. 2D). The requirement forsaturation density also applied to Shh response in NIH-3T3 cellstransiently transfected with luciferase reporter (not shown), and toreporter activation by expression of activated Smo (FIG. 2D), but not toinduction by Gli1 overexpression (not shown).

The steroidal nature of these plant teratogens and their ability todisrupt cholesterol synthesis and/or transport (P. A. Beachy et al.,Cold Spring Harb Symp Quant Biol 62, 191 (1997); Y. Lange, J. Ye, M.Rigney, T. L. Steck, J Lipid Res 40, 2264 (1999)) suggested thepossibility that they may affect the action of Ptc1, which contains anapparent sterol sensing domain (S. K. Loftus et al., Science 277, 232(1997)). Having established the characteristics of Shh response andcyclopamine inhibition in mouse embryonic fibroblasts, we assayedfibroblasts derived from Ptc1−/− mouse embryos for sensitivity tocyclopamine. Mice lacking function of Ptc1 display widespread activationof targets of Shh signaling, including of the Ptc1 gene itself (L. V.Goodrich, L. Milenkovic, K. M. Higgins, M. P. Scott, Science 277, 1109(1997)). As β-galactosidase is expressed under the control of the Ptc1promoter in these cells, β-galactosidase expression can be used to assaythe state of Shh pathway activity (FIG. 3A). Surprisingly, addition ofcyclopamine to Ptc1^(−/−) cells significantly suppressed β-galactosidaseexpression (FIG. 3A) and similarly suppressed activity of the Gli-Lucreporter (not shown), indicating that cyclopamine is able to suppressShh pathway activity in the absence of Ptc1 function. In contrast,cyclopamine failed to prevent pathway activation induced by Gli2overexpression (Table 1). These results suggest that the target ofcyclopamine action is not Ptc1 (Ptc2 is not a likely target ofcyclopamine in Ptc1−/− cells, as expression of the Ptc2 proteinsuppresses pathway activation and the pathway is maximally activated inPtc1−/− fibroblasts (data not shown)), but likely another pathwaycomponent that functions somewhere between Ptc1 and the Gli proteins.

To further investigate the site of cyclopamine action we transfectedNIH-3T3 cells with Smo cDNA (a mouse Smo cDNA probe was generated usingRT-PCR with degenerate oligonucleotide primers based on rat and humanSmo sequences; this probe was subsequently used to isolate a cDNA clonecontaining the complete coding sequence of mouse Smo), and found thatoverexpression of Smo in the absence of Shh induces reporter expression˜10-fold. As this activation of the pathway occurs in the absence of Shhand can be suppressed by 5 μM cyclopamine (FIG. 3B), we infer that themechanism of cyclopamine action is not direct interference with Shhbinding (i.e., as a neutral antagonist of Shh). Interestingly,cyclopamine at this concentration showed little effect on reporterexpression induced by the tumor-derived activated Smo mutants (FIG.21B), suggesting the possibility that cyclopamine acts directly orindirectly upon Smo and that activating mutations render Smo proteinsresistant. Cyclopamine resistance of SmoA1 also was observed atsub-maximal levels of pathway activation associated with reduced SmoA1expression (FIG. 3B). In addition, we tested whether Shh signalingthrough activated Smo is affected by cyclopamine. Although activated Smoproteins previously have been reported to resist suppression by Ptc1 (M.Murone, A. Rosenthal, F. J. de Sauvage, Current Biol. 9, 76 (1999)) wefound that this resistance is partial, as transfection of a 9 to 1 ratioof a Ptc1 construct (not shown) or of Ptc1-CTD, a C-terminally deletedconstruct (Ptc1-CTD previously was shown to be expressed at higherlevels than Ptc1; N. Fuse et al., Proc Natl Acad Sci USA 96, 10992(1999)), can completely inhibit the activating effects of SmoA1 or SmoA2(FIG. 3C). In cells thus transfected, the Gli-responsive reporter can beinduced upon treatment with ShhN_(p); induction under thesecircumstances is resistant to 5 μM cyclopamine (FIG. 3C), which normallywould abolish Shh signaling. These results indicate that activated Smomolecules in the presence of sufficiently high levels of Ptc1 cancontribute to an essentially normal, albeit cyclopamine resistant,response to the Shh signal. The requirement for higher levels of Ptc1 isnot due simply to a higher level of the Smo protein variant, as we foundthat the levels of wild type and activated Smo proteins produced intransfected cells were similar, despite dramatically elevated levels ofreporter activity associated with activated Smo (FIG. 3D).

Although increasing levels of cyclopamine produce some inhibition ofactivated Smo (although little if any inhibitory effect on activated Smois observed at 3 μM cyclopamine (not shown), a concentration sufficientto completely inhibit normal Shh signaling, some inhibitory effect of 5μM cyclopamine is demonstrated in FIG. 4B), complete inhibition wasprecluded by the toxic effects of cyclopamine that emerge in the 10-40μM range (data not shown). However, a chemically synthesized cyclopaminederivative, 3-keto, N-aminoethyl aminocaproyl dihydrocinnamoylcyclopamine (KAAD cyclopamine, compound 33), displayed 10-20 foldgreater potency in suppression of ShhN_(p)-induced pathway activitywhile maintaining similar or lower toxicity (FIG. 4A). This compoundsuppressed SmoA1-induced reporter activity at a concentrationapproximately 10-fold higher than that required for suppression ofShhN_(p) signaling (FIG. 4A) and also displayed higher potency thancyclopamine in p2^(PTC−/−) cells (FIG. 4B). This more potent cyclopaminederivative thus suppresses activated Smo as effectively as high levelsof Ptc1.

The simplest explanation of cyclopamine resistance as conferred byactivated Smo proteins is that cyclopamine affects Smo activity and thatactivating mutations render Smo proteins resistant. An alternativeinterpretation would be that activated Smo proteins produce a highabundance of a downstream component and that a high cyclopamine level isrequired to suppress the increased concentration of this downstreamcomponent. This alternative model, however, can not account for thesustained cyclopamine resistance of activated Smo proteins observed atintermediate or low levels of pathway activation (FIG. 3B) (productionby activated Smo of high levels of a downstream component that is thecyclopamine target can not explain the sustained cyclopamine resistanceobserved at intermediate levels of pathway activation (FIG. 3B), as thehypothetical cyclopamine target in this circumstance would be present atthe same moderate levels as those produced by ShhN_(P) signaling viaunaltered Smo. We also find that high levels of Smo inmaximally-stimulated cells do not confer cyclopamine resistance (notshown), again inconsistent with the notion that extensive production oractivation by Smo of a downstream component can confer cyclopamineresistance). As activated Smo is not expressed at higher levels thanunaltered Smo (FIG. 3D), it would appear that activating mutations mayconfer a higher intrinsic ability to activate the pathway. This suggeststhat, like other seven transmembrane receptors (R. A. Bond et al.,Nature 374, 272 (1995); H. R. Bourne, Curr Opin Cell Biol 9, 134(1997)), Smo may exist in a balance between active and inactive forms.Cyclopamine and Ptc activities might shift this balance toward theinactive state and tumor-associated mutations toward the active state,thus accounting for the higher levels of Ptc and cyclopamine activityrequired to suppress activated Smo proteins.

Cyclopamine appears to impact the Shh pathway at the level of Smoactivity (see above), but this action need not be direct and couldoperate through an effect on molecules involved in intracellulartransport, on molecules that affect posttranslational modification ofSmo, or on other molecules that impact Smo activity. Such indirectaction of cyclopamine would not necessarily be inconsistent with aconformational transition between active and inactive Smo, asconformational state could be coupled by a variety of mechanisms tosubcellular localization or state of covalent modification. Whatever themechanism, such inhibitors may have utility in treatment of disorderscaused by inappropriate Shh pathway activation. Patients with Basal CellNevus Syndrome (also termed Gorlin's syndrome), an autosomal dominantdisorder associated with heterozygous loss-of-function mutations in thehuman Ptc1 gene, display increased incidence of numerous tumors, mostnotably basal cell carcinoma (BCC), medulloblastoma, rhabdomyosarcomaand fibrosarcoma. Loss-of-function mutations in Ptc1 or activatingmutations in Smo in addition are found in ˜40% of sporadic BCC and ˜25%of primitive neuroectodermal tumors. The ability of cyclopamine and itsderivatives to block pathway activation by both of these types ofmutations suggests that these plant-derived compounds or others thatinfluence the activity of Smo may be valuable as therapeutic agents.

Example 2 Steroid Derivatives

New Derivatives Synthesized

Cyclopamine and jervine (structures 1 and 2 of FIG. 5, respectively) areclosely related plant-derived steroidal alkaloids known to specificallyinhibit the Sonic hedgehog signaling pathway (Cooper et al., Science280, p. 1603-1607, 1998). We have synthesized chemically 23 newderivatives of these two compounds, by various modifications of itssecondary amine, the C-3 oxygen, and/or the C5-C6 olefin. Some of thesecompounds can be readily synthesized in labelled form, thus making themuseful for binding studies. Some of these compounds also containfunctional groups useful for photo-activatable cross-linking andconsequent radiolabelling or attachment of a biotin moiety to targetproteins. One of the compounds is fluorescently labelled, and may beuseful for direct observation of the cellular target of cyclopamineaction. The potency of the various derivatives is set forth in Table II.

TABLE II Compound IC₅₀ 1 >100 nM 2 >100 nM 3 >1 μM 12 >10 μM 18 >100 nM19 >10 nM 20 >100 nM 21 >10 nM 22 >1 μM 26 >100 nM 29 >100 nM 30 >100 nM33 >10 nM 34 >10 nM 39 >100 nM 40 >100 nM 41 >100 nM 42 >100 nM 43 >100nM 50 >1 μM 51 >1 μM 57 >10 μM 58 >10 μMRealization of Improved Potencies Through SAR Studies

Using a clonal cell line derived from parental NIH-3T3 fibroblast cellsthat contains a stably integrated Shh-responsive luciferase reporter, wehave determined the concentration of each compound required to achieve50% inhibition of Sonic hedgehog induction (IC50). Through SAR studieswe have found that adducts to the 3,β-hydroxyl dramatically reduceactivity, whereas oxidation of the 3,β-hydroxyl to a keto groupincreases potency. We have also found that addition of bulky groups tothe secondary amine reduces potency, but that longer aliphatic linkersnot only permit addition of such bulky groups but also enhance potency.The most active compound thus far identified (structure 34 in FIG. 5;IC50=30 nM) displays a potency ten-fold greater than that of cyclopamine(structure 1; IC50=300 nM). It should be straighforward to achieve evengreater potencies by systematically testing various adducts to thesecondary amine and combining these in combination with a 3-ketofunctionality. The more potent derivatives we have already synthesizeddisplay the desirable property of achieving inhibition of Shh signalingwith much reduced toxicity as compared to the parent compounds.

Broad Utility of Compounds

We have determined that these compounds are capable of blocking pathwayactivity in cells with elevated levels of pathway activity due to lackof function of the Patched1 (Ptc1) protein, or to constitutivelyactivated function of the Smo protein. The ability of these compounds toblock pathway activity in cells with both of these types of defectssuggests that they may be broadly useful in the treatment of certainsporadic tumors or in prophylactic treatment of patients with aninherited disposition to high frequency formation of these tumors. Suchtumors include but are not limited to basal cell carcinoma,medulloblastoma, fibrosarcoma, and rhabdomyosarcoma. Additionalapplications for these compounds include but are not limited toinduction of pancreatic tissue, elimination of excessive hair growth,and treatment of other skin disorders.

Response to the Hh signal is controlled by two transmembrane proteins,Patched (Ptc) and Smoothened (Smo). Ptc is a twelve-span transmembraneprotein that binds directly to Hh. Smo contains seven transmembranespans, is related to the Frizzled family of Wnt receptors, and isrelated more distantly to members of the G-protein coupled receptorfamily. Genetic evidence indicates that Ptc suppresses the activity ofSmo; Hh relieves this suppression and allows activation of downstreamtargets, including the Ci/GLI family of transcriptional effectors.

The Ptc1 protein is involved in suppressing pathway activity, and cellslacking it display constitutively high levels of pathway activation. Alack of Ptc1 function is causally associated with a high percentage ofsporadic basal cell carcinoma, medulloblastoma, fibrosarcoma,rhabdomyosarcoma, as well as other tumors. In addition familialheterozygosity at the Ptc1 locus is associated with a predisposition tohigh frequency formation of these tumors. We have shown thatcyclopamine, jervine, and related compounds are capable of fullysuppressing pathway activity in cells lacking Ptc function.

The Smo protein is required for pathway activation, and certainmutations of the Smo locus result in constitutive pathway activity, evenin the absence of Shh stimulation. We have found that cells expressingsuch activated Smo proteins are somewhat resistant to these compounds,but can still be suppressed at levels between one and two orders ofmagnitude higher than those required for suppression of normal cellsstimulated with Shh protein. Thus, tumors associated with sporadicactivating mutations at the Smo locus may also be responsive totreatment with these compounds.

Summary of Structure-Activity-Relationship (SAR) Data.

Modifications to cyclopamine (1) and jervine (2) are most efficientlyaccomplished at heteroatom positions, namely the secondary amine and thesecondary alcohol. Our initial investigations of alkaloid derivativestherefore focused upon the chemical synthesis of such heteroatommodifications and the biological evaluation of these novel compounds.Using standard synthetic procedures and a cell-based Shh signalingassay, it was determined that the conjugation of chemical groups to thesteroid alcohol through a carbamate linkage effectively diminishes Shhinhibitory activity; C3-OH modified compounds 8 and 12 exhibit IC50s inthe cell-based assay that are nearly 100 times higher than those ofcyclopamine. These carbamate-containing compounds are also 10 timesweaker than cycloposine (3), which has a more labile modification of theC3-OH group, suggesting that the observed inhibitory activity ofcycloposine is actually due to partial hydrolysis of the glycosidiclinkage.

In contrast, certain modifications of the secondary amine that preservethe basicity of this moiety are accommodated by the cyclopamine target.All N-alkyl derivatives in this study were synthetically obtainedthrough the diamine intermediate 17, which allows for the efficientpreparation of numerous cyclopamine analogs. Even medium-sizedstructural elements such as those found in compounds 29, 33, 39, 40, and43 do not significantly diminish the abilities of these alkaloids toblock Shh signaling, and most additions even appear to accentuateinhibitory activity (29, 33, 39, and 43). These observations aresomewhat different from those reported by Keeler and co-workers, inwhich small N-alkyl derivatives of cyclopamine demonstrated diminishedteratogenic potential in live animals. The reasons for these differencesare unclear, although they could reflect metabolic and/or pharmacologicinfluences in the animal-based studies. There are still limitations,however, on the type of N-alkyl structures that are accepted by thecyclopamine target, according to our cell-based assays. Compounds withlarge steric bulk are unable to block Shh signaling at concentrations upto 15 μM (derivatives 57, 58, and 63), either due to cell membraneimpermeability or steric exclusion from the cyclopamine-interactingsite. Even relatively small, branched elements close to the cyclopamineskeleton significantly abrogate biological activity (compound 50).

These compounds illustrate the variety of N-modifications that areaccepted by the cyclopamine target. It should be noted, however, thatoxidation of the hydroxyl group to a ketone consistently improves theinhibitory activities of the cyclopamine derivatives by approximatelytwo-fold (see compound 18 vs. 19; 20 vs. 21; 22 vs. 26; 33 vs. 34; 40vs. 41; and 50 vs. 51). Formation of the enone by migration of the C5-C6olefin to the C4-C5 position (see compounds 26 and 41) does not appearto affect compound potency, suggesting that the importance ofunsaturation at the C₅-C₆ position reported by Keeler and co-workers isprimarily due to sp²-hybridation at the C5 carbon. Collectively, theseSAR data have facilitated the synthesis of potent Shh signalinginhibitors (for example, compound 34 is the most potent Shh inhibitorknown to date), radiolabeled-probes (30 and 39), photoaffinity reagents(39 and 41), and fluorophores (43).

Most significantly, the compounds described in this study exemplify thefacility by which derivatives of cyclopamine can be synthesized andevaluated. The versatile intermediate 17 should promote the developmentof further cyclopamine-based molecules, expediting the discovery ofderivatives with desirable pharmacological properties. Such cyclopamineanalogs could also be rapidly prepared through combinatorial syntheticapproaches; the cyclopamine skeleton of 17 could be immobilized on asolid support via the C3-OH, and structurally diverse functionalitiescould be conjugated to the primary amine in a repetitive split-and-poolmanner. In principle, this strategy would permit the simultaneoussynthesis of millions of cyclopamine derivatives in a format amenable tohigh throughput screening.

Preparation of Compounds

General Synthetic Procedures.

All reactions were performed under a positive pressure of nitrogen. Airand moisture sensitive compounds were introduced via syringe or cannulathrough a rubber septum. All reagents and solvents were analytical gradeand used as received with the following exceptions. DMF and DMSO werestored over 4 Å molecular sieves, and water was de-ionized anddistilled. Flash chromatography purifications were performed with theindicated solvent system on Merck silica gel 60 (230-400 mesh). Low andhigh resolution mass spectra were obtained by the Mass SpectrometryFacility at the Harvard Department of Chemistry and Chemical Biology.Proton magnetic resonance spectra (¹H NMR) were recorded on a Varian 500MHz spectrophotometer.

Isolation of Crude Cyclopamine (1)

A benzene extract of Veratrum californicum (6.06 g) obtained from theUnited States Department of Agriculture was purified by flashchromatography (SiO₂, step-wise gradient from 50:1 to 6.25:1dichloromethane/ethanol to yield crude cyclopamine as a light brownsolid (460 mg, approximately 1.12 mmoles). ¹H NMR: spectrum confirms theisolation of cyclopamine along with some impurities.

Isolation of Crude Jervine (2).

A benzene extract of Veratrum virides (1.1 g) obtained from the UnitedStates Department of Agriculture was purified by flash chromatography(SiO₂, step-wise gradient from 50:1 to 6.25:1 dichloromethane/ethanol)to yield a brown oil (550 mg). The residue was then recrystallized inethanol/water (35 mL; 1:1) to yield a slightly yellow solid (215 mg,approximately 505 μmoles). Concentration and recrystallization of themother liquor yielded another batch of crude jervine (101 mg,approximately 237 μmoles) ¹H NMR: spectrum confirms the isolation ofjervine along with some impurities.

Cycloposine (3).

Cycloposine was obtained from the United States Department ofAgriculture as a white solid.

Dihydrocinnamic acid N-Hydroxysuccinimide ester (4)

Dihydrocinnnamoyl chloride (638 μL, 4.21 mmoles) was added dropwise to asolution of N-hydroxysuccinimide (500 mg, 4.21 mmoles) and triethylamine(704 μL, 5.05 mmoles) in dichloromethane (5 mL) at 0° C. The reactionwas warmed to room temperature stirred for 1 h. The reaction mixture wasthen added to diethyl ether (50 mL), washed with 1 N HCl (1×20 mL) andsaturated aqueous NaHCO₃ (1×20 mL), dried over MgSO₄, and concentratedin vacuo to yield a white solid (1.03 g, 4.17 mmoles, 99%). HRMS: (EI+)calcd for C₁₃H₁₃NO₄ (M+H): 247.0845. found: 247.0841. ¹H NMR: spectrumis consistent with the predicted structure.

N-Trifluoroacetyl cyclopamine (5)

Trifluoroacetic anhydride (77.3 μL, 547 μmoles) was added to solution ofcrude cyclopamine (75.0 mg, 182 μmoles) and triethylamine (102 μL, 729μmoles) in dichloromethane (0.5 mL), and the mixture was stirred for 10min at room temperature. The reaction mixture was evaporated to drynessby a stream of nitrogen gas and resuspended in MeOH (2 mL). The methanolsolution was refluxed for 45 min then evaporated to dryness in vacuo.Purification by flash chromatography (SiO₂, step-wise gradient from 8:1to 2:1 hexane:acetone) yielded the amide as a white solid (47.3 mg, 93.2μmoles, 51%). LRMS: (ES+) calcd for C₂₉H₄₀NO₃F₃ (M+H): 508. found: 508.¹H NMR: spectrum is consistent with the predicted structure.

N-Trifluoroacetyl, 3O-Succinimidylcarbonyl Cyclopamine (6)

Disuccinimidyl carbonate (107 mg, 417 μmoles) was added to solution of 5(42.3 mg, 83.3 μmoles) and triethylamine (116 μL, 833 μmoles) inacetonitrile (1.0 mL), and the mixture was stirred for 5 h at roomtemperature. The reaction mixture was dissolved in diethyl ether (10mL), washed with 5% citric acid (1×2 mL) and saturated aqueous NaHCO₃(1×2 mL), dried over MgSO₄, and concentrated in vacuo. Purification byflash chromatography (SiO₂, step-wise gradient from 8:1 to 2:1hexane/acetone) yielded the carbonate as a white solid (35.6 mg, 54.9μmoles, 66%). LRMS: (ES+) calcd for C₃₄H₄₃N₂O₇F₃ (M+H): 649. found: 649.¹H NMR: spectrum is consistent with the predicted structure.

N-Trifluoroacetyl, 3O-dihydrocinnamoylethylenediaminecarbamoylcyclopamine (7)

Ethylenediamine (22.9 μL, 342 μmoles) was added to solution of 6 (11.1mg, 17.1 μmoles) in dichloromethane (0.5 mL), and the mixture wasstirred for 15 min at room temperature. The reaction mixture wasevaporated to dryness by a stream of nitrogen gas and excessethylenediamine was removed in vacuo. The resultant residue wasredissolved in dichloromethane (0.5 mL) and treated with 4 (10.6 mg,4.28 μmoles) and triethylamine (5.97 μL, 42.8 μmoles). After stirring atroom temperature for 30 min, the solution was filtered through a plug ofglass wool. Purification by flash chromatography (SiO₂, step-wisegradient from 4:1 to 1:1 hexane/acetone) yielded the carbamate as awhite solid (6.7 mg, 9.23 μmoles, 54%). LRMS: (ES+) calcd forC₄₁H₅₄N₃O₅F₃ (M+H): 726. found: 726. ¹H NMR: spectrum is consistent withthe predicted structure.

3O-dihydrocinnamoylethylenediaminecarbamoyl cyclopamine (8)

Compound 7 (3.0 mg, 4.13 μmoles) was dissolved in a 2 M solution ofammonia in methanol (0.5 mL, 1.00 mmoles). The reaction was stirred atroom temperature for 2 h and then evaporated to dryness by a stream ofnitrogen gas. Purification by flash chromatrography (SiO₂, step-wisegradient from 20:1:0.1 to 20:2:0.1 chloroform/methanol/triethylamine)yielded the amine as a colorless oil (2.1 mg, 3.33 μmoles, 81%). LRMS:(ES+) calcd for C₃₉H₅₅N₃O₄ (M+H): 630. found: 630. ¹H NMR: spectrum isconsistent with the predicted structure.

N-Trifluoroacetyl jervine (9)

Trifluoroacetic anhydride (84.4 μL, 299 μmoles) was added to solution ofcrude jervine (50.9 mg, 120 μmoles) and triethylamine (100 μL, 359μmoles) in dichloromethane (0.5 mL), and the mixture was stirred for 15min at room temperature. The reaction mixture was evaporated to drynessby a stream of nitrogen gas and resuspended in MeOH (0.5 mL). Themethanol solution was stirred at room temperature for 10 min thenevaporated to dryness in vacuo. Purification by flash chromatography(SiO₂, step-wise gradient from 16:1 to 2:1 hexane:acetone) yielded theamide as a white solid (37.8 mg, 72.5 μmoles, 60%). LRMS: (ES+) calcdfor C₂₉H₃₈NO₄F₃ (M+H): 522. found: 522. ¹H NMR: spectrum is consistentwith the predicted structure.

N-Trifluoroacetyl, 3O-succinimidylcarbonyl jervine (10)

Disuccinimidyl carbonate (92.9 mg, 363 μmoles) was added to solution of9 (37.8 mg, 72.5 μmoles) and triethylamine (101 μL, 725 μmoles) inacetonitrile (1.0 mL), and the mixture was stirred for 6 h at roomtemperature. The reaction mixture was dissolved in diethyl ether (10mL), washed with 5% citric acid (1×2 mL) and saturated aqueous NaHCO₃(1×2 mL), dried over MgSO₄, and concentrated in vacuo. Purification byflash chromatography (SiO₂, step-wise gradient from 8:1 to 2:1hexane/acetone) yielded the carbonate as a white solid (38.4 mg, 57.9μmoles, 80%). HRMS: (ES+) calcd for C₃₄H₄₁N₂O₈F₃ (M+Na): 685.2712.found: 685.2711. ¹H NMR: spectrum is consistent with the predictedstructure.

N-Trifluoroacetyl, 3O-dihydrocinnamoylethylenediaminecarbamoyl jervine(11)

Ethylenediamine (20.2 μL, 302 μmoles) was added to solution of 10 (10.0mg, 15.1 μmoles) in dichloromethane (0.5 mL), and the mixture wasstirred for 15 min at room temperature. The reaction mixture wasevaporated to dryness by a stream of nitrogen gas and excessethylenediamine was removed in vacuo. The resultant residue wasredissolved in dichloromethane (0.5 mL) and treated with 4 (7.47 mg,30.2 μmoles) and triethylamine (4.21 μL, 30.2 μmoles) at 0° C. Afterstirring at 0° C. for 30 min, the solution was filtered through a plugof glass wool. Purification by flash chromatography (SiO₂, step-wisegradient from 4:1 to 1:1 hexane/acetone) yielded the carbamate as awhite solid (8.0 mg, 10.8 μmoles, 72%). LRMS: (ES+) calcd forC₄₁H₅₂N₃O₆F₃ (M+H): 740. found: 740. ¹H NMR: spectrum is consistent withthe predicted structure.

3O-dihydrocinnamoylethylenediaminecarbamoyl jervine (12)

Aqueous ammonia (200 μL of a 29% (w/w) solution in water, 3.04 mmoles)was added to a solution of 11 (1.0 mg, 1.35 μmoles) in methanol (200μL). The reaction was stirred at room temperature for 30 min and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatrography (SiO₂, step-wise gradient from 20:1:0.1 to 20:2:0.1chloroform/methanol/triethylamine) yielded the amine as a colorless oil(0.8 mg, 1.24 μmoles, 92%). LRMS: (ES+) calcd for C₃₉H₅₃N₃O₅ (M+H): 644.found: 644. ¹H NMR: spectrum is consistent with the predicted structure.

N-Trifluoroacetyl glycine (13)

Methyl trifluoroacetate (804 μL, 7.99 mmoles) and triethylamine (928 μL,6.66 mmoles) were added to a suspension of glycine (500 mg, 6.66 mmoles)in methanol (2.5 mL). After the mixture was stirred vigorously for 18 h,1 N HCl was added dropwise until the a pH of 2 was obtained. Thereaction was added to ethyl acetate (30 mL) was washed with 1 N HCl(2×10 mL), dried over MgSO₄, and concentrated in vacuo to yield theamide as a white solid (991 mg, 5.79 mmoles, 87%). LRMS: (CI+) calcd forC₄H₄NO₃F₃ (M+NH₄): 189. found: 189. ¹H NMR: spectrum is consistent withthe predicted structure.

N-Trifluoroacetyl glycine N-hydrosuccinimide ester (14)

Disuccinimidyl carbonate (300 mg, 1.17 mmoles) was added to a solutionof 13 (200 mg, 1.17 mmoles) and pyridine (94.6 μL, 1.17 mmoles) inacetonitrile (1.0 mL). The reaction mixture was stirred at roomtemperature for 3 h, during which the solution became clear and evolvedgas. The solution was added to ethyl acetate (10 mL), washed with 1N HCl(2×5 mL) and saturated aqueous NaHCO₃ (2×5 mL), dried over MgSO₄, andconcentrated in vacuo to yield a white solid (232 mg, 865 μmoles, 74%).LRMS: (ES+) calcd for C₈H₇N₂O₅F₃ (M+NH₄): 286. found: 286. ¹H NMR:spectrum is consistent with the predicted structure.

N—(N′-Trifluoroacetyl glycyl)cyclopamine (15)

Triethylamine (135 μL, 972 μmoles) and 14 (261 mg, 972 μmoles) wereadded to a solution of cyclopamine in dichloromethane (2.0 mL). Thereaction was stirred at room temperature for 1 h and then subjecteddirectly to purification by flash chromatrography (SiO₂, step-wisegradient from 8:1 to 2:1 hexane/acetone) yielded the amide as a whitesolid (166 mg, 294 μmoles, 60%). LRMS: (ES+) calcd for C₃₁H₄₃N₂O₄F₃(M+H): 565. found: 565. ¹H NMR: spectrum is consistent with thepredicted structure.

N-Glycyl cyclopamine (16)

Aqueous ammonia (3 mL of a 29% (w/w) solution in water, 45.6 mmoles) wasadded to a solution of 15 (162 mg, 296 μmoles) in methanol (4 mL). Thereaction was stirred at room temperature for 5 h and then evaporated todryness in vacuo. Purification by flash chromatrography (SiO₂;chloroform, then step-wise gradient from 20:1:0.1 to 20:2:0.1chloroform/methanol/triethylamine) yielded the amine as a white solid(110 mg, 235 μmoles, 79%). LRMS: (ES+) calcd for C₂₉H₄₄N₂O₃ (M+H): 469.found: 469. ¹H NMR: spectrum is consistent with the predicted structure.

N-Aminoethyl cyclopamine (17)

Lithium aluminum hydride (939 μL of a 1 M solution in THF, 939 μmoles)was added to a suspension of 16 (110 mg, 235 μmoles) in THF (6 mL). Thereaction was refluxed for 3 h and then quenched with water (5 mL) andaqueous KOH (10 mL of a 10% solution). After extracting the mixture withchloroform (2×20 mL), the organic layer was dried over Na₂SO₄, filtered,and concentrated in vacuo. Purification by flash chromatography (SiO₂,step-wise gradient from 20:1:0.1 to 20:2:0.1chloroform/methanol/triethylamine) yielded the diamine as a colorlessoil (94.4 mg, 208 μmoles, 88%). LRMS: (ES+) calcd for C₂₉H₄₆N₂O₂ (M+H):455. found: 455. ¹H NMR: spectrum is consistent with the predictedstructure.

N—(N′-Dihydrocinnamoyl aminoethyl)cyclopamine (18)

Triethylamine (5.03 μL, 36.1 μmoles) and 4 (4.46 mg, 18.0 μmoles) wereadded to a solution of 17 (8.2 mg, 18.0 μmoles) in dichloromethane (500μL). The reaction was stirred at room temperature for 3 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 4:1 to 1:1 hexane/acetone)yielded the amide as a white solid (5.7 mg, 9.71 μmoles, 54%). LRMS:(ES+) calcd for C₃₈H₅₄N₂O₃ (M+H): 587. found: 587. ¹H NMR: spectrum isconsistent with the predicted structure.

3-Keto, N—(N′-dihydrocinnamoyl aminoethyl)cyclopamine (19)

Dimethylsulfoxide (6.89 μL, 97.1 μmoles) was added to a solution ofoxalyl chloride (4.24 μL, 48.6 μmoles) in dichloromethane (250 μL) at−78° C. After the mixture was stirred at −78° C. for 10 min, a solutionof 18 (5.7 mg, 9.71 μmoles) in dichloromethane (250 μL) was added, andthe reaction was stirred at −78° C. for another 30 min. The oxidationwas completed by the addition of triethylamine (20.3 μL, 146 μmoles) tothe solution, which was stirred at −78° C. for 10 min and then allowedto warm to room temperature. The reaction was quenched by the additionof water (1 mL) and chloroform (5 mL), and the organic layer wasisolated, washed with brine (1×2 mL), dried over over Na₂SO₄, andconcentrated in vacuo. Purification by flash chromatography (SiO₂,step-wise gradient from 8:1 to 4:1 hexane/acetone) to yielded the ketoneas a white solid (2.0 mg, 3.42 μmoles, 35%). Recovered starting material(1.6 mg, 2.73 μmoles, 28%). LRMS: (ES+) calcd for C₃₈H₅₂N₂O₃ (M+H): 585.found: 585. ¹H NMR: spectrum is consistent with the predicted structure.

N—(N′-(4-Benzoylbenzoyl)aminoethyl)benzophenone (20)

4-Benzoylbenzoic acid N-hydroxysuccinimide ester (8.01 mg, 23.5 μmoles)and triethylamine (6.55 μL, 47.0 μmoles) were added to a solution of 17(10.7 mg, 23.5 μmoles) in dichloromethane (500 μL). The reaction wasstirred at room temperature for 2 h and then evaporated to dryness by astream of nitrogen gas. Purification by flash chromatography (SiO₂,step-wise gradient from 100:1 to 50:1 chloroform/methanol) yielded thebenzophenone as a colorless oil (10.8 mg, 16.3 μmoles, 69%). LRMS: (ES+)calcd for C₄₃H₅₄N₂O₄ (M+H): 663. found: 663. ¹H NMR: spectrum isconsistent with the predicted structure.

3-Keto, N—(N′-(4-benzoylbenzoyl) aminoethyl)cyclopamine (21)

Dimethylsulfoxide (11.6 μL, 163 μmoles) was added to a solution ofoxalyl chloride (7.11 μL, 81.5 μmoles) in dichloromethane (250 μL) at−78° C. After the mixture was stirred at −78° C. for 10 min, a solutionof 20 (10.8 mg, 16.3 μmoles) in dichloromethane (250 μL) was added, andthe reaction was stirred at −78° C. for another 30 min. The oxidationwas completed by the addition of triethylamine (34.1 μL, 245 μmoles) tothe solution, which was stirred at −78° C. for 10 min and then allowedto warm to room temperature. The reaction was quenched by the additionof water (1 mL) and chloroform (5 mL). The resultant organic layer wasthen washed with brine (1×2 mL), dried over over Na₂SO₄, andconcentrated in vacuo. Purification by flash chromatography (SiO₂,step-wise gradient from 8:1 to 2:1 hexane/acetone) yielded the ketone asa white solid (6.3 mg, 9.53 μmoles, 58%). LRMS: (ES+) calcd forC₄₃H₅₂N₂O₄ (M+H): 661. found: 661. ¹H NMR: spectrum is consistent withthe predicted structure.

N—(N′-Azidoiodophenylpropionyl aminoethyl)cyclopamine (22)

Azidoiodophenylpropionyl N-hydroxysuccinimide ester (1.9 mg, 4.18μmoles) and triethylamine (2.34 μL, 16.7 μmoles) were added to asolution of 17 (1.9 mg, 4.18 μmoles) in dichloromethane (500 μL). Thereaction was stirred at room temperature for 3 h and then evaporated todryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 4:1 to 1:1 hexane/acetone)yielded the benzophenone as a white solid (1.6 mg, 2.12 μmoles, 51%).LRMS: (ES+) calcd for C₃₈H₅₂N₅O₃I (M+H): 754. found: 754. ¹H NMR:spectrum is consistent with the predicted structure.

N—(N′-Trifluoroacetyl aminoethyl)cyclopamine (23)

Trifluoroacetic anhydride (20.6 μL, 146 μmoles) and triethylamine (24.5μL, 176 μmoles) were added to a solution of 17 (13.3 mg, 29.3 μmoles) indichloromethane (0.5 mL). The mixture was stirred for 30 min at roomtemperature and then evaporated to dryness by a stream of nitrogen gasThe resultant residue was redissolved in methanol (1 mL) and thesolution was stirred at room temperature for 1 h. After removal of thesolvent in vacuo, purification by flash chromatography (SiO₂, step-wisegradient from 4:1 to 2:1 hexane:acetone) yielded the amide as a whitesolid (9.2 mg, 16.7 μmoles, 57%). LRMS: not performed. ¹H NMR: spectrumis consistent with the predicted structure.

3-Keto, N—(N′-trifluoroacetyl aminoethyl)cyclopamine (24)

Dimethylsulfoxide (11.9 μL, 167 μmoles) was added to a solution ofoxalyl chloride (7.28 μL, 83.5 μmoles) in dichloromethane (250 μL) at−78° C. After the mixture was stirred at −78° C. for 10 min, a solutionof 23 (9.2 mg, 16.7 μmoles) in dichloromethane (250 μL) was added, andthe reaction was stirred at −78° C. for another 30 min. The oxidationwas completed by the addition of triethylamine (35.0 μL, 251 μmoles) tothe solution, which was stirred at −78° C. for 10 min and then allowedto warm to room temperature. The reaction was quenched by the additionof saturated aqueous NaHCO₃ (2 mL) and chloroform (5 mL), and theorganic layer was isolated, dried over Na₂SO₄, and concentrated invacuo. Purification by flash chromatography (SiO₂, step-wise gradientfrom 8:1 to 4:1 hexane/acetone) yielded the ketone as a white solid (6.0mg, 10.9 μmoles, 65%). LRMS: not performed. ¹H NMR: spectrum isconsistent with the predicted structure.

3-Enone, N-aminoethyl cyclopamine (25)

Aqueous ammonia (250 μL of a 29% (w/w) solution in water, 3.80 mmoles)was added to a solution of 24 (3.0 mg, 5.47 μmoles) in methanol (250μL). The reaction was stirred at room temperature for 24 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatrography (SiO₂, step-wise gradient from 20:1:0.1 to 20:2:0.1chloroform/methanol/triethylamine) yielded the amine as a colorless oil(3.0 mg, 6.63 μmoles, quant.). LRMS: not performed. ¹H NMR: spectrum isconsistent with the predicted structure.

3-Enone, N—(N′-azidoiodophenylpropionyl aminoethyl)cyclopamine (26)

Azidoiodophenylpropionyl N-hydroxysuccinimide ester (1.4 mg, 3.31μmoles) and triethylamine (1.8 μL, 13.2 μmoles) were added to a solutionof 25 (1.5 mg, 3.31 μmoles) in dichloromethane (250 μL). The reactionwas stirred at room temperature for 3 h and then quenched withdimethylaminopropylamine. Purification by flash chromatography (SiO₂,step-wise gradient from 8:1 to 2:1 hexane/acetone) to yield the arylazide as a white solid (0.5 mg, 0.665 μmoles, 20%). LRMS: (ES+) calcdfor C₃₈H₅₀N₅O₃I (M+H): 752. found: 752. ¹H NMR: spectrum is consistentwith the predicted structure.

N-Trifluoroacetyl 12-aminododecanoic acid (27)

Methyl trifluoroacetate (200 μL, 1.99 mmoles) and triethylamine (184 μL,1.32 mmoles) were added to a suspension of 12-aminododecanoic acid (300mg, 1.32 mmoles) in methanol (2 mL). After the mixture was stirredvigorously for 18 h, 1 N HCl was added dropwise until the a pH of 2 wasobtained. The reaction was added to ethyl acetate (20 mL) was washedwith 1 N HCl (2×5 mL), dried over MgSO₄, and concentrated in vacuo toyield the amide as a white solid (398 mg, 1.28 mmoles, 97%). LRMS: notperformed. ¹H NMR: spectrum is consistent with the predicted structure.

N-Trifluoroacetyl 12-aminododecanoic acid N-hydroxysuccinimide ester(28)

Disuccinimidyl carbonate (247 mg, 964 μmoles) was added to a solution of27 (200 mg, 642 μmoles) and pyridine (104 μL, 1.28 mmoles) inacetonitrile (2.0 mL). The reaction mixture was stirred at roomtemperature for 4.5 h, during which the solution became clear andevolved gas. The solution was added to ethyl acetate (10 mL), washedwith 1 N HCl (2×1 mL) and saturated aqueous NaHCO₃ (2×1 mL), dried overMgSO₄, and concentrated in vacuo to yield a white solid (257 mg, 629μmoles, 98%). LRMS: not performed. ¹H NMR: spectrum is consistent withthe predicted structure.

N—(N′—(N″-Trifluoroacetyl aminododecanoyl) aminoethyl)cyclopamine (29)

Triethylamine (4.78 μL, 34.3 μmoles) and 28 (8.4 mg, 20.6 μmoles) wereadded to a solution of 17 (7.8 mg, 17.2 μmoles) in dichloromethane (250μL). The reaction was stirred at room temperature for 12 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 100:1 to 25:1chloroform/methanol) yielded the amide as a white solid (8.0 mg, 10.7μmoles, 62%). LRMS: (ES+) calcd for C₄₃H₆₈N₃O₄ (M+H): 748. found: 748.¹H NMR: spectrum is consistent with the predicted structure.

N—(N′-Propionyl aminoethyl)cyclopamine (30)

Propionyl N-hydroxysuccinimide ester (1.08 mg, 6.33 μmoles) andtriethylamine (1.48 μL, 10.6 μmoles) were added to a solution of 17 (2.4mg, 5.28 μmoles) in dichloromethane (250 μL). The reaction was stirredat room temperature for 12 h and then quenched withdimethylaminopropylamine. Purification by flash chromatography (SiO₂,step-wise gradient from 100:1 to 25:1 chloroform/methanol) yielded theamide as a colorless oil (1.8 mg, 3.52 μmoles, 67%). LRMS: (ES+) calcdfor C₃₂H₅₀N₂O₃ (M+H): 511. found: 511. ¹H NMR: spectrum is consistentwith the predicted structure.

Preparation of ³H-labeled 30.

Propionyl N-hydroxysuccinimide ester (1 mCi, specific activity=100Ci/mmol, 10 nmoles) in ethyl acetate (1.0 mL) was mixed with 17 (1.1 mg,2.5 μmoles) in chloroform (100 μL). The reaction mixture was incubatedwithout stirring for 20 h at room temperature. Purification by flashchromatography (SiO₂, step-wise gradient from 100:1 to 25:1chloroform/methanol) yielded the tritium-labeled cyclopamine derivative.Fractions containing the desired product were pooled and concentratedwith a stream of nitrogen gas. The concentration solution wasresuspended in methanol (200 μL) and stored at −20° C.Beta/scintillation counter analysis determined the reaction yield to beapproximately 81%, and thin layer chromatography analysis (Rf=0.80;10:2:0.1 dichloromethane/methanol/triethylamine) is consistent withknown properties of cold 30.

N-Dihydrocinnamoyl aminocaproic acid (31)

Aminocaproic acid (100 mg, 747 μmoles) and 4 (185 mg, 747 μmoles) weredissolved in DMF/water (1 mL; 1:1). The reaction was stirred at roomtemperature for 1 h, and then acidified with 1 N HCl until a pH of 2 wasobtained. The mixture was added to ethyl acetate (10 mL), washed with 1N HCl (2×5 mL), dried over MgSO4, and concentrated in vacuo to yield awhite waxy solid (175 mg, 665 μmoles, 89%). LRMS: not performed. ¹H NMR:spectrum is consistent with the predicted structure.

N-Dihydrocinnamoyl aminocaproic acid N-hydroxysuccinimide ester (32)

Disuccinimidyl carbonate (155 mg, 604 μmoles) was added to a solution of31 (159 mg, 604 μmoles) and pyridine (97.7 μL, 1.21 mmoles) inacetonitrile (1 mL). The reaction mixture was stirred at roomtemperature for 2.5 h, during which the solution became clear andevolved gas. The solution was added to ethyl acetate (5 mL), washed with1 N HCl (2×1 mL) and saturated aqueous NaHCO₃ (2×1 mL), dried overMgSO₄, and concentrated in vacuo to yield a colorless oil (156 mg, 433μmoles, 72%). LRMS: not performed. ¹H NMR: spectrum is consistent withthe predicted structure.

N—(N′—(N″-Dihydrocinnamoyl aminocaproyl)aminoethyl)cyclopamine (33)

Triethylamine (4.43 μL, 31.8 μmoles) and 32 (5.75 mg, 15.9 μmoles) wereadded a solution of 17 (7.25 mg, 15.9 μmoles) in dichloromethane (250μL). The reaction was stirred at room temperature for 1 h and evaporatedto dryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 100:1 to 25:1chloroform/methanol) yielded the amide as a colorless oil (5.8 mg, 8.29μmoles, 52%). LRMS: (ES+) calcd for C₄₄H₆₅N₃O₄ (M+H): 700. found: 700.¹H NMR: spectrum is consistent with the predicted structure.

3-Keto, N—(N′—(N″-dihydrocinnamoyl aminocaproyl)aminoethyl)cyclopamine(34)

Dimethylsulfoxide (12.7 μL, 177 μmoles) was added to a solution ofoxalyl chloride (7.73 μL, 88.6 μmoles) in dichloromethane (250 μL) at−78° C. After the mixture was stirred at −78° C. for 10 min, a solutionof 33 (6.2 mg, 8.86 μmoles) in dichloromethane (250 μL) was added, andthe reaction was stirred at −78° C. for another 30 min. The oxidationwas completed by the addition of triethylamine (37.1 μL, 266 μmoles) tothe solution, which was stirred at −78° C. for 10 min and then allowedto warm to room temperature. The reaction was quenched by the additionof saturated aqueous NaHCO₃ (2 mL) and extracted with chloroform (2×2mL). The resultant organic layer was then isolated, dried over overNa₂SO₄, and concentrated in vacuo. Purification by flash chromatography(SiO₂, step-wise gradient from 100:1 to 25:1 chloroform/methanol) toyielded the ketone as a slightly yellow oil (5.4 mg, 7.74 μmoles, 87%).LRMS: (ES+) calcd for C₄₄H₆₃N₃O₄ (M+H): 698. found: 698. ¹H NMR:spectrum is consistent with the predicted structure.

N-Trifluoroacetyl aminocaproic acid (35)

Methyl trifluoroacetate (513 μL, 5.10 mmoles) and triethylamine (474 μL,3.40 mmoles) were added to a suspension of aminocaproic acid (455 mg,3.40 mmoles) in methanol (2 mL). After the mixture was stirredvigorously for 8 h, 1 N HCl was added dropwise until the a pH of 2 wasobtained. The reaction was added to ethyl acetate (10 mL) was washedwith 1 N HCl (2×2 mL), dried over MgSO₄, and concentrated in vacuo toyield the amide as a white solid (745 mg, 3.49 mmoles, quant.). LRMS:not performed. ¹H NMR: spectrum is consistent with the predictedstructure.

N-Trifluoroacetyl aminocaproic acid N-hydroxysuccinimide ester (36)

Disuccinimidyl carbonate (541 mg, 2.11 mmoles) was added to a solutionof 35 (300 mg, 1.41 mmoles) and pyridine (227 μL, 2.81 mmoles) inacetonitrile (2.0 mL). The reaction mixture was stirred at roomtemperature for 13 h, during which the solution became clear and evolvedgas. The solution was added to ethyl acetate (10 mL), washed with 1 NHCl (2×1 mL) and saturated aqueous NaHCO₃ (2×1 mL), dried over MgSO₄,and concentrated in vacuo to yield a white solid (471 mg, 1.45 mmoles,quant.). LRMS: not performed. ¹H NMR: spectrum is consistent with thepredicted structure.

N—(N′—(N″-Trifluoroacetyl aminocaproyl)aminoethyl)cyclopamine (37)

Triethylamine (12.3 μL, 88.0 μmoles) and 36 (17.1 mg, 52.8 μmoles) wereadded to a solution of 17 (20.0 mg, 44.0 μmoles) in dichloromethane (200μL). The reaction mixture was stirred at room temperature for 13 h,quenched with dimethylaminopropylamine (11.2 μL, 88.0 μmoles), andevaporated to dryness with a stream of nitrogen gas. Purification byflash chromatography (SiO₂, step-wise gradient from 50:1 to 25:1chloroform/methanol) yielded the amide as a colorless oil (26.9 mg, 40.5μmoles, 92%). LRMS: not performed. ¹H NMR: spectrum is consistent withthe predicted structure.

N—(N′-Aminocaproyl aminoethyl)cyclopamine (38)

Aqueous ammonia (200 μL of a 29% (w/w) solution in water, 3.04 mmoles)was added to a solution of 37 (26.9 mg, 40.5 μmoles) in methanol (400μL). The reaction was stirred at room temperature for 20 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatrography (SiO₂, step-wise gradient from 20:1:0.1 to 20:4:0.1chloroform/methanol/triethylamine) yielded the amine as a white waxysolid (19.3 mg, 34.0 μmoles, 84%.). LRMS: not performed. ¹H NMR:spectrum is consistent with the predicted structure.

N—(N′—(N″-Azidoiodophenylpropionyl aminocaproyl)aminoethyl)cyclopamine(39)

Azidoiodophenylpropionyl N-hydroxysuccinimide ester (1.4 mg, 3.38μmoles) and triethylamine (0.94 μL, 6.76 μmoles) were added to asolution of 38 (1.92 mg, 3.38 μmoles) in dichloromethane (250 μL). Thereaction was stirred at room temperature for 2.5 h and evaporated todryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 4:1:0.025 to 1:2:0.015hexane/acetone/triethylamine) yielded the azide as a colorless oil (2.2mg, 2.54 μmoles, 75%). LRMS: (ES+) calcd for C₄₄H₆₃N₆O₄I (M+H): 867.found: 867. ¹H NMR: spectrum is consistent with the predicted structure.

Preparation of ¹²⁵I-labeled 39.

¹²⁵I-labeled azidoiodophenylpropionyl N-hydroxysuccinimide ester (0.250mCi, specific activity=2200 Ci/mmol, 0.114 nmoles) in ethyl acetate (2.1mL) was concentrated to a volume of approximately 10 μL by a stream ofnitrogen gas. The concentrated solution was diluted with ethyl acetate(100 μL) and was mixed with 38 (1.0 mg, 1.76 μmoles) in chloroform (100μL). The reaction was mixture was incubated without stirring for 43 h atroom temperature and then concentrated to approximately 10 μL by astream of nitrogen gas. The residue was resuspended in chloroform (200μL) and purified by flash chromatography (SiO₂, step-wise gradient from100:1 to 12.5:1 chloroform/methanol) to yield the radiolabeled azide.Fractions containing the desired product were pooled, concentrated by astream of nitrogen gas, resuspended in methanol (1 mL), and a smallaliquot removed for quantitation. Gamma counter analysis determined thereaction yield to be essentially quantitative, and the solution wasreconcentrated by a stream of nitrogen gas, resuspended in methanol (250μL), and stored at −20° C. Thin layer chromatography analysis (Rf=0.62;10:2:0.1 dichloromethane/methanol/triethylamine) is consistent withknown properties of cold 39.

N—(N′—(N″-Biotinoyl aminocaproyl)aminoethyl)cyclopamine (40)

Biotinoyl N-hydroxysuccinimide ester (6.72 mg, 14.8 μmoles) andtriethylamine (3.43 μL, 24.6 μmoles) were added to a solution of 38 (5.6mg, 12.3 μmoles) in DMF (250 μL). The reaction was stirred at roomtemperature for 14 h and then added to chloroform (2 mL). The organicmixture was washed with saturated aqueous NaHCO₃ (3×1 mL), dried overNa₂SO₄, and concentrated in vacuo. Purification by flash chromatography(SiO₂, step-wise gradient from 20:1:0.05 to 20:5:0.05chloroform/methanol/triethylamine) yielded the ketone as a white solid(9.4 mg, 12.5 μmoles, quant.). LRMS: (ES+) calcd for C₄₅H₇₁N₅O₅S (M+H):794. found: 794. ¹H NMR: spectrum is consistent with the predictedstructure.

3-Enone, N—(N′—(N″-Biotinoyl aminocaproyl)aminoethyl)cyclopamine (41)

Dimethylsulfoxide (3.66 μL, 51.6 μmoles) was added to a solution ofoxalyl chloride (2.25 μL, 25.8 μmoles) in dichloromethane (250 μL) at−78° C. After the mixture was stirred at −78° C. for 10 min, a solutionof 40 (4.1 mg, 5.16 μmoles) in dichloromethane (200 μL) was added, andthe reaction was stirred at −78° C. for another 30 min. The oxidationwas completed by the addition of triethylamine (10.8 μL, 77.4 μmoles) tothe solution, which was stirred at −78° C. for 10 min and then allowedto warm to room temperature. The reaction was quenched by the additionof saturated aqueous NaHCO₃ (2 mL) and extracted with chloroform (2×2mL). The resultant organic layer was then dried over over Na₂SO₄, andconcentrated in vacuo. The residue was redissolved in MeOH (0.5 mL) andtreated with aqueous ammonia (250 μL of a 29% (w/w) solution in water,3.80 mmoles) for 20 h at room temperature. The reaction was added tochloroform (2 mL), washed with saturated aqueous NaHCO₃ (2×2 mL), driedover Na₂SO₄, and concentrated in vacuo. Purification by flashchromatography (SiO₂, step-wise gradient from 20:1:0.1 to 20:5:0.1chloroform/methanol/triethylamine) to yielded the enone as a yellowishsolid (1.4 mg, 1.77 μmoles, 34%). LRMS: (ES+) calcd for C₄₅H₆₉N₅O₅S(M+H): 792. found: 792. ¹H NMR: spectrum is consistent with thepredicted structure.

N—(N′—(N″-Propionyl aminocaproyl)aminoethyl)cyclopamine (42)

Propionyl N-hydroxysuccinimide ester (1.00 mg, 5.87 μmoles) andtriethylamine (1.37 μL, 9.80 μmoles) were added to a solution of 38(2.78 mg, 4.90 μmoles) in dichloromethane (250 μL). The reaction wasstirred at room temperature for 12 h and then quenched withdimethylaminopropylamine. Purification by flash chromatography (SiO₂,step-wise gradient from 100:1 to 10:1 chloroform/methanol) yielded theamide as a colorless oil (2.5 mg, 4.01 μmoles, 82%). LRMS: (ES+) calcdfor C₃₈H₆₁N₃O₄ (M+H): 624. found: 624. ¹H NMR: spectrum is consistentwith the predicted structure.

N—(N′—(N″-BODIPY FL aminocaproyl)aminoethyl)cyclopamine (43)

BODIPY FL N-hydroxysuccinimide ester (2.0 mg, 5.28 μmoles) andtriethylamine (0.98 μL, 7.04 μmoles) were added to a solution of 38 (2.0mg, 3.52 μmoles) in dichloromethane (500 μL). The reaction was stirredat room temperature for 20 h and then evaporated to dryness with astream of nitrogen gas. Purification by flash chromatography (SiO₂,step-wise gradient from 50:1 to 12.5:1 chloroform/methanol) yielded thefluorophore as a colorless oil (2.6 mg, 3.09 μmoles, 88%). LRMS: (ES+)calcd for C₄₉H₇₀N₅O₄BF₂ (M+H): 842. found: 842. ¹H NMR: spectrum isconsistent with the predicted structure.

N-Trifluoroacetyl 4-benzoylphenylalanine (45)

Methyl trifluoroacetate (89.6 μL, 891 μmoles) and triethylamine (104 μL,743 μmoles) were added to a suspension of 4-benzoylphenylalanine (200mg, 743 μmoles) in methanol (0.5 mL). The reaction was stirredvigorously for 24 h and then acidified with 1 N HCl until a pH of 2 wasobtained. The mixture was added to ethyl acetate (10 mL), washed with 1N HCl (2×10 mL), dried over MgSO₄, and concentrated in vacuo to yield awhite solid (49.7 mg, 136 μmoles, 18%). LRMS: not performed. ¹H NMR:spectrum is consistent with the predicted structure.

N-Trifluoroacetyl 4-benzoylphenylalanine N-hydroxysuccinimide ester (46)

Disuccinimidyl carbonate (33.0 mg, 129 μmoles) was added to a solutionof 45 (47.1 mg, 129 μmoles) and pyridine (20.9 μL, 258 μmoles) inacetonitrile (0.5 mL). The reaction mixture was stirred at roomtemperature for 3 h, during which the solution became clear and evolvedgas. The solution was added to ethyl acetate (5 mL), washed with 1 N HCl(2×1 mL) and saturated aqueous NaHCO₃ (2×1 mL), dried over MgSO₄, andconcentrated in vacuo to yield a white solid (48.8 mg, 106 μmoles, 82%).LRMS: not performed. ¹H NMR: spectrum is consistent with the predictedstructure.

Levulinic Acid N-Hydroxysuccinimide Ester (47)

Disuccinimidyl carbonate (692 mg, 2.70 mmoles) and pyridine (437 μL,2.70 mmoles) was added to a solution of levulinic acid (320 mg, 2.70mmoles) in acetonitrile (2.0 mL). The reaction mixture was stirred atroom temperature for 4.5 h, during which the solution became clear andevolved gas. The solution was added to ethyl acetate (10 mL), washedwith 1N HCl (2×5 mL) and saturated aqueous NaHCO₃ (2×5 mL), dried overMgSO₄, and concentrated in vacuo to yield a white solid (333 mg, 1.56mmoles, 58%). LRMS: not performed. ¹H NMR: spectrum is consistent withthe predicted structure.

N—(N′—(N″-Trifluoroacetyl 4-benzoylphenylalanine) aminoethyl)cyclopamine(48)

Triethylamine (12.6 μL, 90.6 μmoles) and 46 (20.9 mg, 45.3 μmoles) wereadded to a solution of 17 (20.6 mg, 45.3 μmoles) in dichloromethane (0.5mL). The reaction was stirred at room temperature for 1 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 8:1 to 1:1 hexane/acetone)yielded the benzephenone as a white solid (22.7 mg, 28.3 μmoles, 62%).LRMS: not performed. ¹H NMR: spectrum is consistent with the predictedstructure.

N—(N′-(4-benzoylphenylalanine) aminoethyl)cyclopamine (49)

Aqueous ammonia (0.5 mL of a 29% (w/w) solution in water, 7.6 mmoles)was added to a solution of 48 (20.4 mg, 25.4 μmoles) in methanol (1 mL).The reaction was stirred at room temperature for 19 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 40:1:0.1 to 40:2:0.1chloroform/methanol/triethylamine) yielded the amine as a colorless oil(17.9 mg, 25.4 μmoles, quant.). LRMS: not performed. ¹H NMR: spectrum isconsistent with the predicted structure.

N—(N′—(N″-Levulinoyl(4-benzoylphenylalaminoyl))aminoethyl)cyclopamine(50)

Triethylamine (5.30 μL, 38.0 μmoles) and 47 (8.10 mg, 38.0 μmoles) wereadded to a solution of 49 (13.4 mg, 19.0 μmoles) in dichloromethane (0.5mL). The reaction was stirred at room temperature for 4 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatography (SiO₂, step-wise gradient from 40:1:0 to 40:2:0.1chloroform/methanol/triethylamine) yielded the diketone as a colorlessoil (11.3 mg, 14.1 μmoles, quant.). LRMS: (ES+) calcd for C₅₀H₆₅N₃O₆(M+H): 804. found: 804. ¹H NMR: spectrum is consistent with thepredicted structure.

3-Keto N—(N′—(N″-Levulinoyl(4-benzoylphenylalaminoyl))aminoethyl)cyclopamine (51)

Dimethylsulfoxide (12.9 μL, 182 μmoles) was added to a solution ofoxalyl chloride (7.96 μL, 91.2 μmoles) in dichloromethane (250 μL) at−78° C. After the mixture was stirred at −78° C. for 10 min, a solutionof 50 (5.65 mg, 7.03 μmoles) in dichloromethane (250 μL) was added, andthe reaction was stirred at −78° C. for another 30 min. The oxidationwas completed by the addition of triethylamine (38.1 μL, 273 μmoles) tothe solution, which was stirred at −78° C. for 10 min and then allowedto warm to room temperature. The reaction was quenched by the additionof saturated aqueous NaHCO₃ (2 mL) was extracted with chloroform (2×5mL). The resultant organic layer was then dried over Na₂SO₄, andconcentrated in vacuo. Purification by flash chromatography (SiO₂,step-wise gradient from 4:1 to 1:1 hexane/acetone) yielded the ketone asa white solid (1.4 mg, 1.75 μmoles, 25%). LRMS: (ES+) calcd forC₅₀H₆₃N₃O₆ (M+H): 802. found: 802. ¹H NMR: spectrum is consistent withthe predicted structure.

N,N′-(4-Benzoylbenzoyl)(tert-butoxycarbonyl)lysine (52)

4-Benzoylbenzoic acid N-hydroxysuccinimide ester (50.0 mg, 147 μmoles)was added to a solution of tert-butoxycarbonyl lysine (43.4 mg, 176μmoles) in DMF (0.5 mL). 1 N NaOH (176 μL, 176 μmoles) and water (74 μL)was added to the reaction, and the mixture was stirred for 4 h at roomtemperature. 1 N NaOH was added again (352 μL, 352 μmoles) and thesolution was stirred overnight. The reaction mixture was then mixed withwater (2 mL) and washed with diethyl ether (2×2 mL). The aqueous layerwas acidified with 1 N HCl until a pH of 2 was obtained, and thesolution was extracted with ethyl acetate (2×2 mL). The resultantorganic layer was dried over MgSO₄ and concentrated in vacuo.Purification by flash chromatography (SiO₂, step-wise gradient from20:1:0.2 to 20:2:0.2 chloroform/methanol/acetic acid) yielded thebenzophenone as colorless oil (40.0 mg, 88.0 μmoles, 60%). LRMS: (ES+)calcd for C₂₅H₃₀N₂O₆ (M+H): 455. found: 455. ¹H NMR: spectrum isconsistent with the predicted structure.

N,N′-(4-Benzoylbenzoyl)(trifluoroacetyl)lysine (53)

Benzophenone 52 (3.55 mg, 78.1 μmoles) was dissolved in trifluoroaceticacid (0.5 mL, 6.49 mmoles), and the solution was stirred at roomtemperature for 1 h. The trifluoroacetic acid was then removed with astream of nitrogen gas, and the residue was resuspended in methanol (0.5mL). After triethylamine (134.8 μL, 968 μmoles) and methyltrifluoroacetate (24.3 μL, 242 μmoles) were added to the methanolsolution, the reaction was stirred for 26 h at room temperature. Thereaction was evaporated to dryness in vacuo and purification by flashchromatography (SiO₂, step-wise gradient from 20:1:0.1 to 20:2:0.1chloroform/methanol/acetic acid) yielded the trifluoroacetamide as acolorless oil (31.3 mg, 69.5 μmoles, 89%). LRMS: (ES+) calcd forC₂₂H₂₁N₂O₅F₃ (M+H): 451. found: 451. ¹H NMR: spectrum is consistent withthe predicted structure.

N,N′-(4-Benzoylbenzoyl)(trifluoroacetyl)lysine N-hydroxysuccinimideester (54)

Disuccinimidyl carbonate (16.0 mg, 62.6 μmoles) was added to a solutionof 53 (28.2 mg, 62.6 μmoles) and pyridine (10.1 μL, 125 μmoles) inacetonitrile (1.0 mL). The reaction mixture was stirred at roomtemperature for 3 h, during which the solution became clear and evolvedgas. The solution was added to ethyl acetate (10 mL), washed with 1 NHCl (1×2 mL) and saturated aqueous NaHCO₃ (1×2 mL), dried over MgSO₄,and concentrated in vacuo to yield a colorless oil (32.8 mg, 59.9μmoles, 96%). LRMS: (ES+) calcd for C₂₆H₂₄N₃O₇F₃ (M+H): 548. found: 548.¹H NMR: spectrum is consistent with the predicted structure

N—(N′—(N″,N′″-(4-Benzoylbenzoyl)(trifluoroacetyl)lysine)aminoethyl)cyclopamine (55)

Triethylamine (8.36 μL, 60.0 μmoles) was added to a solution of 54 (16.4mg, 30.0 μmoles) in dichloromethane (0.5 mL). The solution became brightyellow, indicating racemization of the amino acid α-carbon. A solutionof 17 (13.6 mg, 30.0 μmoles) in dichloromethane (250 μL) was then addedto the yellow solution. The reaction mixture was stirred for 1 h at roomtemperature, during which it became colorless, and purification by flashchromatography (SiO₂, step-wise gradient from 2:1 to 1:2hexane/acaetone) yielded the cyclopamine derivative as a colorless oil(15.8 mg, 17.8 μmoles, 59%, mixture of diastereomers). LRMS: (ES+) calcdfor C₅₁H₆₅N₄O₆F₃ (M+H): 887. found: 887. ¹H NMR: spectrum is consistentwith the predicted structure.

N—(N′-(4-Benzoylbenzoyl)lysine)aminoethyl)cyclopamine (56)

Compound 55 (13.0 mg, 14.7 μmoles) was dissolved in a 2 M solution ofammonia in methanol (1.0 mL, 2.00 mmoles). The reaction was stirred atroom temperature for 23 h and then evaporated to dryness by a stream ofnitrogen gas. Purification by flash chromatrography (SiO₂, step-wisegradient from 20:1:0.1 to 20:2:0.1 chloroform/methanol/triethylamine)yielded the amine as a white waxy solid (11.0 mg, 13.9 μmoles, mixtureof diastereomers, 95%). LRMS: (ES+) calcd for C₄₉H₆₆N₄O₅ (M+H): 791.found: 791. ¹H NMR: spectrum is consistent with the predicted structure.

N—(N′—(N″,N′″-(4-Benzoylbenzoyl) (N″″-digoxigenin 3-O-methylcarbonylaminocaproyl)lysine)aminoethyl)cyclopamine (57)

Digoxigenin 3-O-methylcarbonyl aminocaproic acid N-hydroxysuccinimideester (5.0 mg, 7.59 μmoles) and triethylamine (1.59 μL, 11.4 μmoles)were added to a solution of 56 (4.5 mg, 5.69 μmoles) in dichloromethane(0.5 mL). After the solution was stirred for 2 h at room temperature,ethylenediamine (10 μL, 150 μmoles) was added and the reaction wasstirred for an addition 15 min at room temperature. Saturated aqueousNaHCO₃ (2 mL) was added to the mixture, which was then extracted withchloroform (5 mL, then 2 mL). The organic layers were combined and driedover Na₂SO₄, and concentrated in vacuo. Purification by flashchromatography (SiO₂, step-wise gradient from 20:1 to 5:1dichloromethane/methanol) yielded the digoxigenin derivative as whitewaxy solid (3.5 mg, 2.62 μmoles, 46%). LRMS: (ES+) calcd forC₈₀H₁₁₁N₅O₁₂ (M+H): 1337. found: 1337. ¹H NMR: spectrum is consistentwith the predicted structure.

N—(N′—(N″,N′″-(4-Benzoylbenzoyl)(N″″-biotinoylaminocaproyl)lysine)aminoethyl)cyclopamine (58)

N-Biotinoyl aminocaproic acid N-hydroxysuccinimide ester (2.6 mg, 5.69μmoles) and triethylamine (1.59 μL, 11.4 μmoles) were added to asolution of 56 (4.5 mg, 5.69 μmoles) in DMF (250 μL). After the reactionwas stirred for 2 h at room temperature, ethylenediamine (10 μL, 150μmoles) was added to the solution, which was stirred for an addition 15min at room temperature. Saturated aqueous NaHCO₃ (2 mL) was added tothe mixture, which was then extracted with chloroform (1×2 mL). Theorganic layers were combined and dried over Na₂SO₄, and concentrated invacuo. Purification by flash chromatography (SiO₂, step-wise gradientfrom 20:1 to 5:1 dichloromethane/methanol) yielded the digoxigeninderivative as white waxy solid (3.4 mg, 3.01 μmoles, 53%). LRMS: (ES+)calcd for C₆₅H₉₁N₇O₈S (M+H): 1130. found: 1130. ¹H NMR: spectrum isconsistent with the predicted structure.

N—(N′-Trifluoroacetyl(4-benzoylphenylalanine))aminocaproic acid (59)

A solution of aminocaproic acid (10.8 mg, 80.5 μmoles) in water (100 μL)and a solution of 46 (24.4 mg, 53.7 μmoles) in DMF (100 μL) werecombined and stirred for 45 min at room temperature. The reaction wasthen acidified with 1 N HCl until a pH of 2 was obtained, added to ethylacetate (1 mL), washed with 1 N HCl (2×0.5 mL), dried over MgSO₄, andevaporated to dryness by a stream of nitrogen gas yield a colorless oil(25.9 mg, 54.1 μmoles, quant.). LRMS: not performed. ¹H NMR: spectrum isconsistent with the predicted structure.

N—(N′-Trifluoroacetyl(4-benzoylphenylalanine))aminocaproic acidN-hydroxysuccinimide ester (60)

Disuccinimidyl carbonate (18.7 mg, 73.0 μmoles) was added to a solutionof 59 (23.3 mg, 48.7 μmoles) and pyridine (7.88 μL, 97.4 μmoles) inacetonitrile (200 μL). The reaction mixture was stirred at roomtemperature for 12 h, during which the solution became clear and evolvedgas. The solution was added to ethyl acetate (1 mL), washed with 1 N HCl(1×0.5 mL) and saturated aqueous NaHCO₃ (1×0.5 mL), dried over MgSO₄,and evaporated to dryness by a stream of nitrogen gas yield a colorlessoil (26.4 mg, 45.9 μmoles, 94%.). LRMS: not performed. ¹H NMR: spectrumis consistent with the predicted structure.

N—(N′—(N″—(N′″-Trifluoroacetyl(4-benzoylphenylalaminoyl))aminocaproyl)aminoethyl)cyclopamine(61)

Compound 60 (23.8 mg, 41.4 μmoles) and triethylamine (11.5 μL, 82.8μmoles) were added a solution of 17 (15.7 mg, 34.5 μmoles) indichloromethane (0.5 mL). The reaction was stirred for 1 h at roomtemperature and purification by flash chromatography (SiO₂, step-wisegradient from 2:1 to 1:2 hexane/acetone) yielded the benzophenonederivative as colorless oil (5.3 mg, 5.79 μmoles, 17%). LRMS: notperformed. ¹H NMR: spectrum is consistent with the predicted structure.

N—(N′—(N″-(4-Benzoylphenylalaminoyl) aminocaproyl)aminoethyl)cyclopamine(62)

Aqueous ammonia (250 μL of a 29% (w/w) solution in water, 3.80 mmoles)was added to a solution of 61 (5.3 mg, 5.79 μmoles) in methanol (200μL). The reaction was stirred at room temperature for 18 h and thenevaporated to dryness by a stream of nitrogen gas. Purification by flashchromatrography (SiO₂, step-wise gradient from 20:1:0.05 to 20:2:0.05chloroform/methanol/triethylamine) yielded the amine as a colorless oil(4.0 mg, 4.88 μmoles, 84%). LRMS: not performed ¹H NMR: spectrum isconsistent with the predicted structure.

N—(N′—(N″—(N′″—(N″″-Biotinoylaminocaproyl)(4-benzoylphenylalaminoyl))aminocaproyl)aminoethyl)cyclopamine(63)

N-Biotinoyl aminocaproic acid N-hydroxysuccinimide ester (2.7 mg, 5.86μmoles) and triethylamine (1.4 μL, 9.76 μmoles) were added to a solutionof 62 (4.0 mg, 4.88 μmoles) in DMF (250 μL). After the reaction wasstirred for 15 min at room temperature, it was added to chloroform (2mL), washed with saturated aqueous NaHCO₃, dried over Na₂SO₄, andconcentrated in vacuo. Purification by flash chromatography (SiO₂,step-wise gradient from 20:1:0.05 to 20:4:0.05chloroform/methanol/triethylamine) yielded the biotin derivative aswhite solid (4.5 mg, 3.88 μmoles, 80%). LRMS: (ES+) calcd forC₆₇H₉₅N₇O₈S (M+H): 1158. found: 1158. ¹H NMR: spectrum is consistentwith the predicted structure.

Example 3 In Vivo Testing

Methods:

Female nude mice were injected subcutaneously with 5 million P2A6fibrosarcoma cells derived from Ptc−/− embryonic fibroblasts. Threeweeks after injection, each mouse had developed a discrete subcutaneoustumor. Each tumor was measured and treatments were initiated on the sameday. Mice were treated once daily with intraperitoneal injections oftomatidine, cyclopamine, or KAAD-cyclopamine 33 (one mouse pertreatment) for four days. The mice were killed, and tumors were measuredand dissected out for histopathologic analysis. Tumor volumes werecalculated as the product of length x width. The samples wereparaformaldehyde-fixed and paraffin-embedded, and slides were cut forhematoxylin and eosin staining and for immunohistochemistry withpolyclonal antibodies against the proliferation marker Ki-67 (NovacastraNCL-Ki67p; assay performed according to manufacturer's directions).

Results:

As depicted in the graph, control treatment with tomatidine resulted in17% tumor growth over the treatment period whereas treatment withcyclopamine decreased tumor size by 0.14% and treatment withKAAD-cyclopamine 33 decreased tumor size by 19%.

To confirm these results, Ki-67 proliferation rates were determined by apathologist who was blinded to the treatment conditions. The magentabars in the graph show proliferation rates for the three treatments. Theproliferation rates for tomatidine, cyclopamine, and KAADcyclopamine-treated tumors were 30%, 16%, and 12% respectively.

Since the differentiation of many tumors correlates with prognosis, thedifferentiation status of the tumors was examined. All tumors showedzonal variation in differentiation as illustrated in thephotomicrographs of FIG. 7. Compared to more poorly differentiatedtumors, well differentiated fibrosarcomas have smaller, less crowdednuclei separated by relatively abundant pink collagen. As illustrated,KAAD-cyclopamine and cyclopamine (not shown) treated tumors showed agreater degree of differentiation than did the tomatidine-treatedcontrol.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All patents and publications cited herein are hereby incorporated byreference in their entirety.

We claim:
 1. A method for the treatment of a human subject having abasal cell carcinoma tumor, comprising administering to the subject aneffective amount of a medicament comprising cyclopamine, wherein in saidtumor smoothened-dependent hedgehog signaling is utilized for inhibitionof apoptosis, and wherein said medicament is administered in a dose thatinduces apoptosis in said tumor, causes decrease of size of the tumor,and selectively inhibits smoothened-dependent hedgehog signaling in saidtumor.
 2. A method for the treatment of a human subject having a tumorin which smoothened-dependent hedgehog signaling is used, comprisingadministering to the subject an effective amount of a medicamentcomprising cyclopamine, wherein in said tumor smoothened-dependenthedgehog signaling is utilized for inhibition of apoptosis, and saidmedicament is administered in a dose that induces apoptosis in saidtumor, causes decrease of size of the tumor and selectively inhibitssmoothened-dependent hedgehog signaling in said tumor.
 3. A method ofclaim 2 wherein smoothened-dependent hedgehog signaling is also used forinhibition of differentiation and said dose is sufficient to inducedifferentiation in said tumor.
 4. A method of claim 2 wherein saidamount of cyclopamine is not toxic to the subject.
 5. A method of claim3 wherein said amount of cyclopamine is not toxic to the subject.
 6. Amethod of claim 3 wherein said medicament is a pharmaceuticalcomposition for topical or systemic administration.
 7. A method of claim6 wherein the composition for systemic administration is in the form ofan aqueous solution.
 8. A method of claim 6 wherein the composition issystemically administered by injection.
 9. A method of claim 6 whereinthe pharmaceutical composition achieves controlled release.
 10. A methodof claim 6 wherein the pharmaceutical composition is in the form of apatch or a transdermal patch.
 11. A method of claim 6 wherein thepharmaceutical composition is in the form of a cream or ointment or gelor hydrogel.
 12. A method of claim 2 wherein the tumor is a skin tumorand the medicament is in the form of a pharmaceutical composition forinjection or topical or oral or parenteral administration.
 13. A methodof claim 1 wherein said medicament is in the form of a pharmaceuticalcomposition for topical administration and is applied 4 or more times aday.
 14. A method of claim 13 wherein said medicament is in the form ofa cream.
 15. A method for treatment of a human subject having a tumor,comprising determining that smoothened-dependent hedgehog signaling isutilized for inhibition of apoptosis in said tumor, and administering tothe subject a medicament comprising cyclopamine or a pharmaceuticallyacceptable salt thereof, wherein said medicament is administered in adose that is sufficient to induce apoptosis of said tumor cells andcauses decrease of size of the tumor.
 16. A method of claim 15 whereinsaid tumor is basal cell carcinoma.
 17. A method for the treatment of ahuman subject having a tumor in which smoothened-dependent hedgehogsignaling is used for inhibition of apoptosis and for selectiveinhibition of differentiation in said tumor, comprising administering tothe subject an effective amount of a medicament comprising cyclopamine.18. A method of claim 17 wherein said medicament is a pharmaceuticalcomposition for topical or systemic administration.
 19. A method ofclaim 18 wherein the composition for systemic administration is in theform of an aqueous solution.
 20. A method of claim 18 wherein thecomposition is in the form of a liposome.
 21. A method of claim 18wherein the composition is administered by injection.
 22. A method ofclaim 18 wherein the pharmaceutical composition achieves controlledrelease.
 23. A method of claim 18 wherein the pharmaceutical compositionis in the form of a patch or a transdermal patch.
 24. A method of claim18 wherein the pharmaceutical composition is in the form of a cream orointment or gel or hydrogel.
 25. A method of claim 17 wherein the tumoris a skin tumor and the medicament is in the form of a pharmaceuticalcomposition for injection or topical or oral or parenteraladministration.
 26. A method of claim 6 wherein said medicament is inthe form of a cream or a liposome.